# Patent

## Title
Cellular permissivity factor for viruses and uses thereof

## Abstract

The present invention provides methods and compositions related to the generation of host cells permissive for virus growth, particularly Porcine Reproductive and Respiratory Syndrome (PRRS) virus.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. application Ser. No. 12/775,283, field May 6, 2010, now U.S. Pat. 8,058,050, which is a continuation of U.S. application Ser. No. 11/113,751, filed Apr. 25, 2005, now U.S. Pat. 7,754,464, and claims benefit of U.S. Provisional Patent Application Ser. No. 60/565,214, filed Apr. 23, 2004, and 60/634,736, filed Dec. 9, 2004. The complete text and disclosure of the 12/775,283 parent application is incorporated by reference herein as if fully set forth.


FIELD OF THE INVENTION
The present invention provides methods and compositions related to the generation of host cells permissive for virus growth for viruses of the family Asfarviridae and Arteriviridae.
BACKGROUND OF THE INVENTION
Asfarviridae
Asfarviridae is a family of icosohedral enveloped viruses whose genome consists of a single molecule of linear double-stranded DNA of about 150000-190000 nucleotides long. The name of the family is derived from African Swine Fever and Releted Viruses. African Swine Fever Virus (ASFV) is the type species of the Asfivirus genus and is the sole member of the family Recently, porcine CD163 polypeptide has been surmised by implication to be the cellular receptor for African swine fever virus (ASFV) (Sanchez-Torres et al., 2003)
Arteriviridae
Viruses of the family of Arteriviridae includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). The Arterivirus having the greatest economic importance is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
PRRSV
Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases of swine. The syndrome appeared almost simultaneously in North America and in Western Europe in the late 1980s, and has since spread to become endemic in the major swine producing nations of Europe, Asia, and the Americas. The etiologic agent of PRRS is a virus that has been designated PRRS virus or PRRSV. For both European and North American PRRS, the disease is characterized by reproductive failure in sows and gilts (late term abortions, still births, and mummies), high mortality among nursery pigs, and respiratory disease in swine of all ages. The disease has been the subject of recent reviews (Mengeling and Lager, 2000; Murtaugh et al., 2002; Nodelijk, 2002; Plagemann, 2003).
In the pig, PRRSV infection is limited to a subset of cells of the monocyte/macrophage lineage. Fully differentiated porcine alveolar macrophage (PAM) cells are the primary target cells for viral replication (Duan et al., 1997a; Duan et al., 1997b). Immortalization of PAM cells is technically challenging, and when successful, has resulted in cell lines that are not permissive for PRRS virus growth (Weingartl et al., 2002). PRRS virions are specifically bound by macrophages and internalized in clathrin-coated pits by endocytosis. Release from endocytic vesicles requires acidic pH (Nauwynck et al., 1999). Initial binding of virions is mediated by interaction of the viral matrix protein with heparin sulfate glycosaminoglycans (Delputte et al., 2002). Internalization can be facilitated by a 210 or 220 kDa membrane glycoprotein, as incubation of PAM cells with monoclonal antibodies to this polypeptide blocks infection with PRRS virus (Duan et al., 1998; Wissink et al., 2003). The 210 kDa glycoprotein has recently been identified as sialoadhesin, a member of siglec family of sialic acid binding immunoglobulin-like lectins (Pensaert et al., 2003). Transfection of the non-permissive PK-15 (porcine kidney) cell line with porcine sialoadhesin conferred the ability to internalize PRRSV particles, but there remained an apparent block at the uncoating stage, as virions entered into cellular vesicles but did not undergo nucleocapsid disintegration and vesicle membrane fusion. Viral genes were not expressed, and the transfected PK-15 cells were not rendered permissive for the PRRS virus (Vanderheijden et al., 2003). To our knowledge, transfection with sialoadhesin has not been shown to be sufficient to convert any PRRSV non-permissive cell line to a PRRSV-permissive phenotype.
Apart from primary porcine cells of the monocyte/macrophage lineage, the only other cell type known to be permissive for the growth of PRRSV in cell culture is the immortalized monkey kidney cell line MA-104(Chladek et al., 1998) and derivatives such as MARC-145 (Kim et al., 1993) and CL-2621. It is not known why this one particular cell line is permissive, yet other mammalian cell lines are not. The PRRS virus binds specifically to a number of different cell types, but does not initiate infection (Kreutz, 1998; Therrien et al., 2000). In MARC-145 cells, the internalization of the virus by endocytosis and subsequent uncoating in low pH vesicles seems to mimic similar events in PAM cells (Kreutz and Ackermann, 1996). However, a number of monoclonal antibodies that bind to porcine sialoadhesin fail to detect a homologous protein on the surface of MARC-145 cells (Duan et al., 1998; Wissink et al., 2003), suggesting that MARC-145 cells may use a divergent member of the same protein family or a different receptor altogether.
Current PRRSV vaccines are propagated on simian cell lines, which is a potentially dangerous activity. The use of simian cell lines for vaccine production has the potential to introduce primate viruses of significance into swine lines intended for xenotransplant purposes. Because swine are being increasingly explored as a source of xenotransplanted organs for humans, the introduction of primate cell lines to swine populations may ultimately pose a risk to humans receiving xenotransplanted organs. Thus, it would be prudent to avoid the use of simian cell lines in swine vaccine preparations. It would be therefore desirable to identify or generate non-simian cells or cell lines capable of supporting PRRSV replication. Towards this goal, it is essential to identify the gene product(s) which may be responsible for conferring the permissivity for PRRSV replication as seen in certain simian cells lines as well as PAM cells. Once such gene products are identified, non-permissive cells might be rendered permissive by, for example, transfection of the essential gene into them, thereby affording a wider array of production lines for a vaccine.
One lab has reported that the tetraspanin protein CD151 from MARC-145 cells, when transfected into non-permissive BHK-21 cells, confers permissivity to the PRRS virus (Kapil and Shanmukhappa, 2003; Shanmukhappa and Kapil, 2001). This observation has yet to be confirmed by an independent lab.
We describe here an unrelated polypeptide, which when introduced into non-permissive cells, confers permissivity to the PRRS virus.
References Cited


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SUMMARY OF THE INVENTION
The invention includes a method of facilitating infection of one or more cells by a virus that is selected from the group consisting of Arteriviridae and Asfarviridae, which comprises the step of increasing expression of a CD163 polypeptide within said cell. In a preferred embodiment the CD163 is membrane bound. In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
Increased expression of a CD163 polypeptide may be accomplished by methods such as introduction of exogenous nucleic acids encoding CD163 polypeptides. Such methods include but are not limited to transfection, electroporation and fusion with a carrier of a polynucleotide comprising a polynucleotide encoding a CD163 polypeptide. Increased expression may also be accomplished by induction of expression of endogenous CD163 by chemical treatment.
The method may render previously non PRRSV-permissive cells to be PRRSV permissive. The method may render a cell that was previously PRRSV permissive to be more PRRSV permissive. The method also includes rendering one or more cells that previously did not express a CD163 polypeptide into cells that are induced to express a CD163 polypeptide. The method also includes rendering one or more cells that previously expressed a CD163 polypeptide to express a higher level of CD163 polypeptide.
The cells in a preferred embodiment are animal cells. They may be vertebrate or invertebrate cells. The cells may be mammalian. The cells or cell line may be an insect cell line. The cells may be BHK21 cells. The cells may be derived from porcine kidney cells. The cells or cell line may be derived from feline kidney cells. The cells or cell line may be but are not limited to BHK-21, NLST-1, NLFK-1, Vero, swine testicle, or rabbit lung cells. The cells or cell line may be derived from avian cells. The PRRSV may be of the European or North American genotype.
As noted above, increased expression of a CD163 polypeptide may be accomplished by methods which include but are not limited to: transfection, electroporation and fusion with a carrier of a polynucleotide comprising a polynucleotide encoding a CD163 polypeptide. In addition, viral vectors may be used to introduce a polynucleotide encoding a CD163 polypeptide. Any CD163 polypeptides are contemplated. Those containing a transmembrane region are preferred. Exemplary polynucleotide encoding CD163 polypeptides that may be used in the method are selected from the group consisting of the polynucleotides listed below.
One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 2 by no more than 20 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 2 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 1.

    
    
        One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 70% identity to a polypeptide set forth in SEQ ID NO: 14 or fragments thereof.
    
    


One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90% identity to a polypeptide set forth in SEQ ID NO: 14 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 99% identity to a polypeptide set forth in SEQ ID NO: 14 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 14 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 14 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 14.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 13.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90% identity to a polypeptide set forth in SEQ ID NO: 19 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 19 by no more than 2 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 19.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 18.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 24 by no more than 2 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 24.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 23.
One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 27 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 27 by no more than 20 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 27 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 27.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 26.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 98% or 99% identity to a polypeptide set forth in SEQ ID NO: 32 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 32 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 32 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 31.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 34 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 34 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 34 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 34.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 33.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 36 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 36 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 36 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 36.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 35.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 42 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 42 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 42 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 42.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 41.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 44 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 44 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 44 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 44.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 43.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 46 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 46 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 46 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 46.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 45.
One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 48 or fragments thereof.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 48 by no more than 15 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 48 by no more than 10 conservative amino acid substitutions.
One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 48.
One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 47.
The method of facilitating infection described above may further comprise the step of producing a culture of virus. The invention also comprises the step of isolating the virus from said culture.
The invention further comprises the culture isolated by the method described above.
The invention further comprises the virus isolated by the method described above.
Any of the methods described above may further comprise the step of producing a PRRSV or other viral vaccine. The vaccine may comprise killed or live attenuated virus. The invention further comprises the vaccines produced by said method.
The invention also comprises a cell or cell line wherein the ability of one or more cells to be infected by a virus selected from the group consisting of Arteriviridae and Asfarviridae has been modified by increasing expression of a CD163 polypeptide within said cells. In another embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
In a preferred embodiment the CD163 polypeptide comprises a transmembrane region. In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
The cell or cell line of the invention may have been previously PRRSV non-permissive and is rendered PRRSV permissive by directing increased expression of a CD163 polypeptide within said cell or cell line. The cell or cell line of the invention may have been previously PRRSV permissive and is rendered to be more PRRSV permissive by directing increased expression of a CD163 polypeptide within said cell or cell line.
The cell or cell line of the invention includes cells or cell lines that did not express a CD163 polypeptide and is induced to express a CD163 polypeptide. The cell or cell line of the invention includes cells or cell lines that previously expressed a CD163 polypeptide and is induced to express a higher level of CD163 polypeptide.
The cells in a preferred embodiment are animal cells. They may be vertebrate or invertebrate cells. The cells may be mammalian. The cell or cell line may be an insect cell or cell line. The cells may be BHK21 cells. The cells may be derived from porcine kidney cells. The cell or cell line may be derived from feline kidney cells. The cells may be, but are not limited to, BHK-21, NLST-1, NLFK-1, Vero, swine testicle (ST), or rabbit lung (RL) cells. The PRRSV may be North American or European.
The invention includes a method for measuring the propensity of a test cell or cell line to allow infection by a virus selected from the group consisting of Arteriviridae and Asfarviridae comprising:

    a) providing a sample containing nucleic acids from the test cell or cell line;
    b) determining the amount of polynucleotide encoding a CD163 polypeptide or its complement in said sample;

wherein an increased amount of polynucleotide encoding a CD163 polypeptide relative to a control sample derived from a control cell or cell line known not to support the growth of said virus indicates a propensity of the test cell or cell line to support the replication of said virus.



In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
The amount of polynucleotide encoding a CD163 polypeptide may be determined by hybridization.
The amount of polynucleotide encoding a CD163 polypeptide may be determined by PCR.
The invention also includes a method for measuring the propensity of a test cell or cell line to allow infection by a virus selected from the group consisting of Arteriviridae and Asfarviridae comprising:

    (a) providing a sample containing polypeptides from the test cell or cell line;
    (b) determining the amount of CD163 polypeptide in said sample;

wherein an increased amount of a CD163 polypeptide relative to a control sample derived from a control cell or cell line known not to support the growth of said virus indicates a propensity of the test cell or cell line to support the replication of said virus.



In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
In one embodiment the determining is accomplished by contacting a CD163 polypeptide with an antibody specific for the CD163 polypeptide, under conditions wherein the antibody binds the CD163 polypeptide.
The invention includes a method for measuring the propensity of a first pig to become infected by a virus selected from the group consisting of Arteriviridae and Asfarviridae comprising:

    a) providing a sample containing nucleic acids from the pig to be tested;
    b) determining the amount of polynucleotide encoding a CD163 polypeptide or its complement in said sample;

wherein an increased amount of polynucleotide encoding a CD163 polypeptide relative to the amount of polynucleotide encoding a CD163 polypeptide in a sample from a second pig indicates a greater propensity of the first pig to be infected by said virus.



In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
In one embodiment the determining is accomplished by hybridization. In another embodiment the determining is accomplished by PCR.
The invention also includes a method for measuring the propensity of a first pig to become infected by a virus selected from the group consisting of Arteriviridae and Asfarviridae comprising:

    (a) providing a sample containing polypeptides from pig to be tested;
    (b) determining the amount of CD163 polypeptide in said sample;

wherein an increased amount of a CD163 polypeptide relative to the amount of CD163 polypeptide in a sample from a second pig indicates a greater propensity of the first pig to be infected by said virus.



In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).
In one embodiment the determining is accomplished by contacting a CD163 polypeptide with an antibody specific for the CD163 polypeptide, under conditions wherein the antibody binds the CD163 polypeptide.
The invention also includes an isolated polypeptide wherein the polypeptide is selected from the group consisting of the polypeptides described below.
Therefore the invention also includes an isolated polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 2 by no more than 20 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 2 by no more than 10 conservative amino acid substitutions.
One such polypeptide comprises SEQ ID NO: 2.
The invention also includes an isolated polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 14 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by no more than 10 conservative amino acid substitutions.
One such polypeptide comprises SEQ ID NO: 14.
The invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 19 or fragments thereof.
The invention also includes an isolated polypeptide differing from SEQ ID NO: 19 by no more than 2 conservative amino acid substitutions.
One such polypeptide comprises SEQ ID NO: 19.
The invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 24 or fragments thereof.
The invention also includes an isolated polypeptide differing from SEQ ID NO: 24 by no more than 2 conservative amino acid substitutions.
One such polypeptide comprises SEQ ID NO: 24.
The invention also includes an isolated polypeptide having at least 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 27.
One such polypeptide is a polypeptide differing from SEQ ID NO: 27 by no more than 20 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 27 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 27.
The invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a polypeptide set forth in SEQ ID NO: 32 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 32.
The invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 34 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 34 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 34 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 34.
The invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 36 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 36 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 36 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 36.
The invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 38 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 38 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 38 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 38.
The invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 40 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 40 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 40 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 40.
The invention also includes an isolated polypeptide having at 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 42 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 42 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 42 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 42.
The invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 44 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 44 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 44 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 44.
The invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 46 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 46 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 46 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 46.
Therefore the invention also includes an isolated polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a polypeptide set forth in SEQ ID NO: 48 or fragments thereof.
One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by no more than 15 conservative amino acid substitutions.
One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by no more than 10 conservative amino acid substitutions.
One such polypeptide is a polypeptide comprising SEQ ID NO: 48.
The invention also includes an isolated CD163 polynucleotide wherein said polynucleotide is selected from the group consisting of the polynucleotides enumerated below.
Therefore the invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NOs: 1 or 5;
    (b) a polynucleotide that encodes a polypeptide that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 2 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 2;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NOs: 12 or 13;
    (b) a polynucleotide that encodes a polypeptide that has at least 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 14 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 14;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NOs: 17 or 18;
    (b) a polynucleotide encoding a polypeptide of SEQ ID NO: 19 or fragments thereof;
    (c) a polynucleotide which is the complement of (a) or (b).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NOs: 22 or 23;
    (b) a polynucleotide that encodes a polypeptide that has at least 99%, 99.9% identity to a polypeptide set forth in SEQ ID NO: 24 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 24;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NOs: 25 or 26;
    (b) a polynucleotide that encodes a polypeptide that has at least 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 27;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NOs: 30 or 31;
    (b) a polynucleotide that encodes a polypeptide that has at least 96%, 96.2%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 32;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 33;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 34;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 35;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 36 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 36;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 37;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 38 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 38;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 39;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 40;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 41;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 42 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 42;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 43;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 44 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 44;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


Therefore the invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 45;
    (b) a polynucleotide that encodes a polypeptide that has at least 85%, 85.5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%; 96%, 97%, 98%; or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 46 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 46;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) polynucleotide sequence set forth in SEQ ID NO: 47;
    (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 48 or fragments thereof;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 49;
    (d) a polynucleotide which is the complement of any of (a), (b), or (c).


Therefore the invention also includes a CD163 polypeptide in which the transmembrane region is deleted.
Therefore the invention also includes a polynucleotide encoding a CD163 polypeptide in which the transmembrane region is deleted.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above.
Brief Description of the Sequence Listings


    SEQ ID NO: 1—cDNA sequence encoding porcine susCD163v1
    SEQ ID NO: 2—predicted amino acid sequence of porcine susCD163v1
    SEQ ID NO: 3—cDNA sequence, Genbank accession number AJ311716
    SEQ ID NO: 4—predicted amino acid sequence derived from Genbank accession number AJ311716
    SEQ ID NO: 5—cDNA sequence of susCD163v1 containing flanking (non-coding) sequence
    SEQ ID NO: 6-11—primer sequences
    SEQ ID NO: 12—cDNA sequence encoding porcine susCD163v2 containing flanking (non-coding) sequence
    SEQ ID NO: 13—cDNA sequence encoding porcine susCD163v2
    SEQ ID NO: 14—predicted amino acid sequence of porcine susCD163v2
    SEQ ID NO: 15-16—primer sequences
    SEQ ID NO: 17—cDNA sequence encoding human CD163v2 containing flanking (non-coding) sequence
    SEQ ID NO: 18—cDNA sequence encoding human CD163v2
    SEQ ID NO: 19—predicted amino acid sequence of human CD163v2
    SEQ ID NO: 20-21—primer sequences
    SEQ ID NO: 22—cDNA sequence encoding murine CD163v2 containing flanking (non-coding) sequence
    SEQ ID NO: 23—cDNA sequence encoding murine CD163v2
    SEQ ID NO: 24—predicted amino acid sequence of murine CD163v2
    SEQ ID NO: 25—cDNA sequence encoding murine CD163v3 containing flanking (non-coding) sequence
    SEQ ID NO: 26—cDNA sequence encoding murine CD163v3
    SEQ ID NO: 27—predicted amino acid sequence of murine CD163v3
    SEQ ID NO: 28-29—primer sequences
    SEQ ID NO: 30—cDNA sequence encoding MARC-145 CD163v2 containing flanking (non-coding) sequence
    SEQ ID NO: 31—cDNA sequence encoding MARC-145 CD163v2
    SEQ ID NO: 32—predicted amino acid sequence of MARC-145 CD163v2
    SEQ ID NO: 33—cDNA sequence encoding Vero cell CD163v2 transcript
    SEQ ID NO: 34—predicted amino acid sequence of Vero cell CD163v2
    SEQ ID NO: 35—cDNA sequence encoding Vero cell CD163v3 transcript
    SEQ ID NO: 36—predicted amino acid sequence of Vero cell CD163v3
    SEQ ID NO: 37—cDNA sequence encoding Vero cell CD163v4 transcript
    SEQ ID NO: 38—predicted amino acid sequence of Vero cell CD163v4
    SEQ ID NO: 39—cDNA sequence encoding Vero cell CD163v5 transcript
    SEQ ID NO: 40—predicted amino acid sequence of Vero cell CD163v5
    SEQ ID NO: 41—cDNA sequence encoding Vero cell CD163v6 transcript
    SEQ ID NO: 42—predicted amino acid sequence of Vero cell CD163v6
    SEQ ID NO: 43—cDNA sequence encoding Vero cell CD163v7 transcript
    SEQ ID NO: 44—predicted amino acid sequence of Vero cell CD163v7
    SEQ ID NO: 45—cDNA sequence encoding canine CD163v2 transcript
    SEQ ID NO: 46—predicted amino acid sequence of canine CD163v2
    SEQ ID NO: 47—cDNA sequence encoding canine CD163v3 transcript
    SEQ ID NO: 48—predicted amino acid sequence of canine CD163v3





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Schematic comparison of susCD163v1 with AJ311716
FIG. 2 Amino acid sequence alignment of susCD163v1 (SEQ ID NO: 2) with AJ311716 (SEQ ID NO: 4)
FIG. 3 Nucleotide sequence alignment of susCD163v1 (SEQ ID NO:1) with AJ311716(SEQ ID NO:3).
FIG. 4 Generation of DNA fragments and ligation to place CD163 directly behind the RSV promoter. Plasmids were digested with either DraIII or DrdI, followed by a blunting reaction with Klenow enzyme. After clean up, the plasmids were digested with NotI. Gel purification yielded DNA fragments that were subsequently ligated utilizing the cohesive NotI termini. Promoters from RSV (pRSV) and SV40 (pSV40) are indicated with arrows.
FIG. 5 Map of pCDNA3.1 Directional V5/His/TOPO cloning vector
FIG. 6 Three BHK/CMV/v1 cell lines, #3, #5, and #12 and a non-permissive BHK cell line were infected with PRRSV isolate P129 and stained with SDOW17-FITC. Panel A shows a non-permissive BHK21 cell clone. Panel B shows BHK/CMV/v1 clone #3. Panel C shows BHK/CMV/v1 clone #5. Panel D shows BHK/CMV/v1 clone #12.
FIG. 7 Three BHK/RSV/v1 cell lines, #2, #3, and #4 were infected with PRRSV isolate P129 and stained with SDOW17-FITC. Panel A shows BHK/RSV/v1 clone #2. Panel B shows BHK/RSV/v1 clone #3. Panel C shows BHK/RSV/v1 clone #4.
FIG. 8 Feline kidney cell lines stably expressing porcine CD163v1, showing PRRSV plaques. Cell lines NLFK-CMV-susCD163v1-G4F and NLFK-CMV-susCD163v1-G4L, both at passage 4, were infected with the P129 isolate of North American PRRSV and incubated for 6 days. Monolayers were fixed with 80% acetone and stained with monoclonal antibody SDOW17-FITC. Phase contrast microscopy (right) shows localized regions of viral CPE (plaques), while FA detection (left) shows co-localized viral nucleocapsid antigen.
FIG. 9 Four FK/RSV/v1 cell lines, #1, #2, #3, and #4 were infected with PRRSV isolate P129 and stained with monoclonal antibody SDOW17-FITC. Panel A shows FK/RSVv1 #1 cell clone. Panel B shows FK/RSV/v1 clone #2. Panel C shows FK/RSV/v1 clone #3. Panel D shows FK/RSV/v1 #4.
FIG. 10 PK-CMV-susCD163v1-A10 cells at passage 19, infected with PRRSV isolate P129. Left: The monolayer was fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid. Right: The same well under bright field illumination, showing cell distribution.
FIG. 11 BHK-CMVScript-susCD163v2-A9 at passage 17 infected with PRRSV isolate P129. The monolayer was fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid.
FIG. 12 Three representative examples of the BHK/RSV/v2 cell lines. The cells were infected with PRRSV isolate P129 and subsequently stained with SDOW17-FITC. Panel A shows cell line BHK/RSV/v2 #1, panel B shows cell line BHK/RSV/v2 #34, and panel C shows cell line BHK/RSV/v2 #47.
FIG. 13 FK-cDNA3.1D-humCD163v2-A6 at passage 15 infected with PRRSV isolate P129. The monolayer was then fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid.
FIG. 14 The amount of progeny PRRSV produced by four recombinant cell lines stably expressing susCD163v1, and by MARC-145 cells, was determined in a growth curve experiment using the NVSL 94-3 isolate of PRRSV. Samples harvested at 12-hour intervals were titrated on MARC-145 cell monolayers.
FIG. 15 Flow cytometry analysis of PRRSV infection in the presence of CD163 specific antibody. BHK-21 cells expressing MARC-145 CD163 from transient transfection were incubated with either CD163 specific antibody or normal goat IgG (NGS) and infected with a GFP-expressing PRRSV. Each data point represents the results of triplicate wells.
FIG. 16 Flow cytometry analysis of PRRSV infection in the presence of CD163 specific antibody. NLFK cells stably expressing human CD163 were incubated with either CD163 specific antibody or normal goat IgG (NGS) and infected with a GFP expressing PRRSV. At 24 hours post infection the percentage of GFP expressing infected cells was determined. Each data point represents the result from a single well of cells.
FIG. 17. Graphical depiction of six alternative splicing variants of CD163 mRNA recovered from Vero cells. The six variants differ in the presence or absence of three exons, designated E6, E105, and E83. Exons E6 and E105 have lengths that are multiples of three, and therefore do not result in a change in reading frame when absent. In contrast, the absence of E83 results in a shifted reading frame and an alternative amino acid sequence at the carboxy terminus of the protein (indicated by a hatched pattern in the figure). The hydrophobic transmembrane (TM) region is encoded within E105.
FIG. 18. PK-RSVScript-susCD163v2 #9 cells infected with PRRSV isolate P129. Undiluted supernatant from PRRSV isolate P201 infected PAMs was used to infect PK-RSVScript-susCD163v2 #9 cells. After two days of incubation the cells were fixed and stained with monoclonal antibody SDOW17 as described in Example 11.
FIG. 19. FK-RSVScript-susCD163v2 #51 cells infected with PRRSV isolate P129. Undiluted supernatant from PRRSV isolate P201 infected PAMs was used to infect FK-RSVScript-susCD163v2 #51 cells. Two days post infection the cells were acetone fixed and stained with monoclonal antibody SDOW17 as described in Example 11.
FIG. 20. Infection of PK-RSVScript-susCD163v2 clone #9 cells with PRRSV isolate P201. Panel A shows a monolayer of cells infected with PRRSV P201 at passage 1, twenty-four hours post infection. Panel B shows a monolayer of cells 2 days post infection with cell free supernatant PRRSV P201 at passage 10.
FIG. 21. NLFK parent cells and one subclone of FK-cDNA3.1D-humCD163v2-A6 were examined for the CD163 expression. Cells were fixed in 80% acetone and reacted with Goat anti-human CD163 (R&D System at 1:200) for one hour following by washing with PBS. For visualization, donkey anti-Goat IgG conjugated with FITC (Biodesign Inc at 1:100) were used. No specific fluorescence was detected in the NLFK parent cells as shown in FIG. 21A. The majority of the FK.A6.A2 subclone showed good fluorescent staining indicating the presence of CD163 (FIG. 21B).



DETAILED DESCRIPTION OF THE INVENTION
General Definitions
Cells and cell lines can be either “virus permissive” or “virus non-permissive”. For example, a cell or cell line that is virus permissive is capable of allowing virus infection, subsequent replication, and virus production. A cell or cell line that is virus non-permissive is incapable of allowing virus infection, subsequent replication, and virus production. A cell line that is already somewhat permissive may be rendered more permissive by the methods of the invention.
Arteriviridae refers to a family of enveloped, positive-stranded RNA viruses belonging to the order Nidovirales. The family includes lactate dehydrogenase-elevating virus (LDV) of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), and PRRSV.
Asfarviridae is a family of icosohedral, enveloped viruses whose genomes consist of single molecules of linear double-stranded DNA about 150000-190000 nucleotides long. The name of the family is derived from African Swine Fever And Releted Viruses. African Swine Fever Virus (ASFV) is the type species of the Asfivirus genus and is the sole member of the family.
The term “PRRSV” or PRRS virus refers to both European and North American PRRS virus genotypes. Within each genotype, isolates typically share 85% or higher nucleotide identity. Between genotypes, however, the level of sequence identity is only about 60%. The North American genotype of PRRSV is exemplified by the prototype isolate VR2332. The genomic sequence of VR2332 is found in Genbank accession number U87392, which is incorporated herein by reference. The European genotype of PRRSV is exemplified by the prototype isolate “Lelystad virus”. The genomic sequence of Lelystad virus is found in Genbank accession number M96262, which is incorporated herein by reference.
The PRRS virus is a member of the family Arteriviridae. The genome of the arteriviruses is single-stranded RNA of positive polarity between 12 and 16 kb in length, capped at the 5′ end and polyadenylated at the 3′ end. Over two-thirds of the genome is dedicated to open reading frames (ORFs) 1a and 1b, which encode the non-structural functions of the virus. ORF1b is an extension of ORF1a, and is the result of a ribosomal frameshift. ORFs 1a and 1b are translated directly from the genomic RNA. These large polypeptide products are cleaved by viral proteases to yield 12 or 13 discrete smaller peptides. The remaining ORFs, which encode viral structural proteins, are expressed from a series of 3′ co-terminal subgenomic RNAs (sgRNAs). The sgRNAs are produced by discontinuous transcription of negative-stranded RNA, such that a common 5′ leader sequence becomes fused to each transcript. The major structural proteins are the nucleocapsid (N, encoded by ORF7), the matrix protein (M, encoded by ORF6), and the major envelope glycoprotein (GP5, encoded by ORF5). The remaining proteins, GP4 (ORF4), GP3 (ORF3), GP2 (ORF2a), and E (ORF2b) are minor structural components of the virion, whose functions have not yet been elucidated. The molecular biology of PRRSV has been the subject of recent review articles (Dea et al., 2000; Meulenberg, 2000; Snijder and Meulenberg, 2001).
As used herein, the term “CD163 polypeptide” means a protein encoded by a mammalian CD163 gene, including allelic variants containing conservative or non-conservative changes. A cDNA sequence that encodes a porcine CD163 polypeptide has been reported (Genbank accession number AJ311716). A murine CD163 polypeptide has also been reported (Genbank access number AF274883), as well as multiple human variants, exemplified by Genbank access numbers AAH151281 and CAA80543. We report herein polynucleotides that encode porcine, human, murine, canine, and african green monkey CD163 polypeptides and which comprise the sequences set forth in SEQ ID NO: 1, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47. A “CD163 polypeptide” is a member of the scavenger receptor cysteine-rich (SRCR) family of transmembrane glycoproteins, and is thought to be expressed exclusively on monocytes and macrophages. One identified role of CD163 is to inhibit oxidative tissue damage following hemolysis by consuming hemoglobin:haptoglobin complexes by endocytosis. The subsequent release of interleukin-10 and synthesis of heme oxygenase-1 results in antiinflammatory and cytoprotective effects (Philippidis et al., 2004; Graversen et al., 2002). The human CD163 gene spans 35 kb on chromosome 12, and consists of 17 exons and 16 introns. A number of isoforms of the CD163 polypeptide, including membrane bound, cytoplasmic and secreted types, are known to be generated by alternative splicing (Ritter et al., 1999). Isoforms that comprise a transmembrane domain are particularly preferred.
A transmembrane domain is characterized by a polypeptide segment of a larger sequence that is exposed on both sides of a membrane. The cytoplasmic and extracellular domains are separated by at least one membrane-spanning segment that traverses the hydrophobic environment of the lipid bilayer. The membrane-spanning segment is composed of amino acid residues with nonpolar side chains, usually in the form of an alpha helix. Segments that contain about 20-30 hydrophobic residues are long enough to span a membrane as an alpha helix, and they can often be identified by means of a hydropathy plot. The predicted transmembrane domain of SEQ ID NOs:2 and 14 are indicated by bolding in the specification. To determine whether other CD163 sequences contain a similar sequence feature is easily determined by inspection of the sequence or hydropathy plots. SEQ ID NOs: 37-40 are representative of variant CD163 proteins which do not contain a transmembrane domain and their encoding nucleic acids.
As used hereinafter, “polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides. Polynucleotide and nucleic acid are used synonymously herein.
As used hereinafter, “polypeptide” refers to any peptide or protein comprising amino acids joined to each other by peptide bonds or modified peptide bonds. “Polypeptide” refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. “Polypeptides” include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications or modified forms include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins-Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Postranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al., “Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “Protein Synthesis: Post-translational Modifications and Aging”, Ann NY Acad Sci (1992) 663:4842).
A “fragment” of a polypeptide as used herein means a segment of at least about 25 consecutive amino acids of a polypeptide of the invention. In another embodiment, a “fragment” as used herein means a segment of at least about 50 consecutive amino acids of a polypeptide of the invention. In another embodiment, a “fragment” as used herein means a segment of at least about 100 consecutive amino acids of a polypeptide of the invention.
As used hereinafter, “isolated” means altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein. Therefore “isolated” as used herein and as understood in the art, whether referring to “isolated” polynucleotides or polypeptides, is taken to mean separated from the original cellular environment in which the polypeptide or nucleic acid is normally found. As used herein therefore, by way of example only, a transgenic animal or a recombinant cell line constructed with a polynucleotide of the invention makes use of the “isolated” nucleic acid. Specifically excluded from the definition of isolated polynucleotides of the invention are entire isolated chromosomes from native host cells from which the polynucleotide was originally derived.
In the disclosure to follow we will often make use of the term “identity” or similarity as applied to the amino acid sequences of polypeptides. Percent amino acid sequence “identity” with respect to polypeptides is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the target sequences after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence identity is determined by conventional methods. For example, BLASTP 2.2.6 [Tatusova TA and TL Madden, “BLAST 2 sequences—a new tool for comparing protein and nucleotide sequences.” (1999) FEMS Microbiol Lett. 174:247-250.]
Briefly, as noted above, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1, and the “blosum62” scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci. USA 89:10915-10919. 1992).
The percent identity is then calculated as:




  
    
      Total
      ⁢
      
        
      
      ⁢
      number
      ⁢
      
        
      
      ⁢
      of
      ⁢
      
        
      
      ⁢
      identical
      ⁢
      
        
      
      ⁢
      matches
    
    
      
        
          
            
              
                
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                      length
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                      ⁢
                      
                        
                          
                        
                        ⁢
                        
                          
                        
                      
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                      ⁢
                      
                        
                      
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                      ⁢
                      
                        
                      
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                      gaps
                    
                  
                
              
            
            
              
                
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                  ⁢
                  
                    
                  
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                  ⁢
                  
                    
                      
                    
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                  ⁢
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              align
              ⁢
              
                
              
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              ⁢
              
                
                  
                
                ⁢
                
                  
                
              
              ⁢
              two
              ⁢
              
                
              
              ⁢
              sequences
            
            ]
          
        
      
    
  
  ×
  100




Percent sequence “similarity” (often referred to as “homology”) with respect to a polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the target sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (as described above), and also considering any conservative substitutions as part of the sequence identity.




  
    
      
        
          
            Total
            ⁢
            
              
            
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            number
            ⁢
            
              
            
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            of
            ⁢
            
              
            
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            identical
            ⁢
            
              
            
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            matches
          
        
      
      
        
          
            and
            ⁢
            
              
            
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            conservative
            ⁢
            
              
            
            ⁢
            substitutions
          
        
      
    
    
      
        
          
            
              
                
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                      ⁢
                      
                        
                      
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              align
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  ×
  100




Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
Exemplary conservative substitutions are set out in Tables 1, 2, and 3 below.







TABLE 1







Conservative Substitutions I










SIDE CHAIN




CHARACTERISTIC
AMINO ACID







Aliphatic




Non-polar
G A P




I L V



 



Polar - uncharged
C S T M




N Q



 



Polar - charged
D E




K R



 



Aromatic
H F W Y



 



Other
N Q D E










Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp. 71-77] as set out in Table 2, immediately below







TABLE 2







Conservative Substitutions II








SIDE CHAIN



CHARACTERISTIC
AMINO ACID





Non-polar (hydrophobic)



A. Aliphatic:
A L I V P


 


B. Aromatic:
F W


 


C. Sulfur-containing:
M


 


D. Borderline:
G


 


Uncharged-polar



A. Hydroxyl:
S T Y


 


B. Amides:
N Q


 


C. Sulfhydryl:
C


 


D. Borderline:
G


 


Positively Charged (Basic):
K R H


 


Negatively Charged (Acidic):
D E









As still another alternative, exemplary conservative substitutions are set out in Table 3, immediately below.







TABLE 3







Conservative Substitutions III










Original Residue
Exemplary Substitution







Ala (A)
Val, Leu, Ile



Arg (R)
Lys, Gln, Asn



Asn (N)
Gln, His, Lys, Arg



Asp (D)
Glu



Cys (C)
Ser



Gln (Q)
Asn



Glu (E)
Asp



His (H)
Asn, Gln, Lys, Arg



Ile (I)
Leu, Val, Met, Ala, Phe,



Leu (L)
Ile, Val, Met, Ala, Phe



Lys (K)
Arg, Gln, Asn



Met (M)
Leu, Phe, Ile



Phe (F)
Leu, Val, Ile, Ala



Pro (P)
Gly



Ser (S)
Thr



Thr (T)
Ser



Trp (W)
Tyr



Tyr (Y)
Trp, Phe, Thr, Ser



Val (V)
Ile, Leu, Met, Phe, Ala










Methods Directed to the Production of Virus and Host Cells of the Invention
The invention provides a method of modifying production of a virus that is a member of the family Arteriviridae and Asfarviridae in a cell comprising the step of directing said cell to express a CD163 polypeptide. This may include rendering a virus non-permissive cell into a virus permissive cell, or may involve rendering a cell more permissive to the virus.
In one embodiment, the virus that is a member of the family Arteriviridae or Asfarviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), PRRSV of swine, and ASFV of swine.
In a preferred embodiment the virus is PPRSV.
The invention further provides a method of preparing a culture of a virus that is a member of the family Arteriviridae or Asfarviridae comprising the steps of providing a cell line; directing said cell line to express a CD163 polypeptide; infecting said cell line with virus; and causing said cell line to produce viral progeny.
In one embodiment, the virus that is a member of the family Arteriviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), PRRSV of swine and ASFV of swine.
In a preferred embodiment the virus is PRRSV.
All of the above methods utilize cells and cell lines expressing a CD163 polypeptide. Expression of CD163 may be facilitated or increased by methods that involve the introduction of exogenous nucleic acid into the cell. Such a cell may comprise a polynucleotide or vector in a manner that permits expression of an encoded CD163 polypeptide.
Polynucleotides that encode CD163 may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein-coding region, or in a viral vector. Methods for introducing exogenous nucleic acid into the host cell are well known and routinely practiced in the art, including transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Host cell systems of the invention include invertebrate and vertebrate cells systems. Hosts may include, but are not limited to, the following: insect cells, porcine kidney (PK) cells, feline kidney (FK) cells, swine testicular (ST) cells, African green monkey kidney cells (MA-104, MARC-145, VERO, and COS cells), Chinese hamster ovary (CHO) cells, baby hampster kidney cells, human 293 cells, and murine 3T3 fibroblasts. Insect host cell culture systems may also be used for the expression of CD163 polypeptides. In another embodiment, the CD163 polypeptides are expressed using a drosophila expression system.
The choice of a suitable expression vector for expression of the CD163 polypeptides will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pSport and pcDNA3 (Invitrogen), pCMV-Script (Stratagene), and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and modifier sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Rous sarcoma virus (RSV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.
Because CD163 sequences are known to exist in cells from various species, the endogenous gene may be modified to permit, or increase, expression of the CD163 polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring CD163 promoter with all or part of a heterologous promoter, so that the cells express CD163 polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous CD163 encoding sequences. [See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.] It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional cad gene, which encodes for carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the CD163 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the CD163 coding sequences in the cells.
CD163 expression may also be induced by chemical treatment. Phorbol esters, especially phorbol myristyl acetate (PMA), activate one or more isozymes of the ubiquitous membrane receptor, protein kinase C (PKC) and are particularly preferred means of increasing CD163 expression. Other methods of intracellular calcium mobilization are also contemplated.
Vaccine Production
The methods described above may be used to produce any virus that is a member of the family Arteriviridae or Asfarviridae for the purpose of vaccine production or diagnostics.
In one embodiment the virus that is a member of the family Arteriviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), and PRRSV of swine.
In a preferred embodiment the virus is PRRSV.
Vaccine Production
The methods described above may be used to produce virus for the purpose of vaccine production or diagnostics.
Killed (inactivated) or live vaccines can be produced. Therefore, to make a live vaccine, a viral isolate, or an attenuated or mutated variant thereof, is grown in cell culture. The virus is harvested according to methods well known in the art. The virus may then be concentrated, frozen, and stored at −70° C., or freeze-dried and stored at 4° C. Prior to vaccination the virus is mixed at an appropriate dosage, (which is from about 103 to 108 tissue culture infectious doses per ml (TCID50/ml)), with a pharmaceutically acceptable carrier such as a saline solution, and optionally an adjuvant.
The vaccine produced might also comprise an inactivated or killed virus comprising a virus grown by the methods of the invention. The inactivated vaccine is made by methods well known in the art. For example, once the virus is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods well known in the art. For example, the virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate viral antigenic mass to produce vaccines. The virus is then inactivated by treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), or other methods known to those skilled in the art. The inactivated virus is then mixed with a pharmaceutically acceptable carrier such as a saline solution, and optionally an adjuvant. Examples of adjuvants include, but not limited to, aluminum hydroxide, oil-in-water and water-in-oil emulsions, AMPHIGEN, saponins such as QuilA, and polypeptide adjuvants including interleukins, interferons, and other cytokines.
Inactivation by formalin is performed by mixing the viral suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%. The virus-formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature. The inactivated virus mixture is then tested for residual live virus by assaying for growth on a suitable cell line.
Inactivation by BEI is performed by mixing the viral suspension of the present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. The virus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours. The inactivated virus mixture is tested for residual live virus by assaying for growth on a suitable cell line.
Virus permissive cells that have been directed to express CD163 can also be used to quantify live virus. Two common methods, which are well known to those skilled in the art, are the plaque assay and the limiting dilution assay.
CD163-expressing cell lines of the present invention can be used to grow virus for the purpose of producing viral antigen for diagnostic kits. For example lysates from infected cells (with optional purification of viral particles or extraction of selected viral proteins) may be coated on ELISA plates in order to detect and quantify antibodies to the virus in swine sera.
Live or inactivated virus grown in CD163-expressing cells can be used after optional separation of the viral proteins to immunize animals in order to generate polyclonal, monospecific or monoclonal antibodies. These in turn can be used as the basis of diagnostic assays for the detection and quantification of virus in swine serum and other biological samples.
Assays of the Invention
The invention provides methods for determining the propensity of an animal to be infected by a virus that is a member of the family Arteriviridae or Asfarviridae, or of a cell line to support the replication of a virus that is a member of the family Arteriviridae or Asfarviridae. Samples from either source are obtained and assayed for expression of CD163. The level of CD163 gene expression can be compared with levels of controls known not to support replication of the virus, where an increased amount of CD163 gene expression relative to levels from the controls indicates a greater propensity of the non-control to be infected by said virus.
In the case of an animal, samples can be any sample comprising sample nucleic acid molecules or proteins, and obtained from any bodily tissue expressing CD163, including, but not limited to, alveolar macrophages, cultured cells, biopsies, or other tissue preparations. The level of expression can be assessed at the level of genomic DNA, messenger RNA, and/or protein produced. In a preferred embodiment the member of the virus of the family Arteriviridae or Asfarviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), PRRSV of swine, and ASFV of swine.
Nucleic Acid Based Assays
Methods of determining CD163 levels may be nucleic acid based as noted above. CD163-derived nucleic acids may be in solution or on a solid support. In some embodiments, they may be employed as array elements in microarrays alone or in combination with other array element molecules. Nucleic acid based methods generally require the isolation of DNA or RNA from the sample and subsequent hybridization or PCR amplification using specific primers derived from any known CD163 encoding sequences in the art or those specifically disclosed as SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47. DNA or RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Tijssen, P. (1993) Laboratory Techniques in Biochemistry and Molecular Biology Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, New York, N.Y. In one preferred embodiment, total RNA is isolated using the TRIZOL total RNA isolation reagent (Life Technologies, Inc., Gaithersburg Md.) and mRNA is isolated using oligo d(T) column chromatography or glass beads. When sample nucleic acid molecules are amplified it is desirable to amplify the sample nucleic acid molecules and maintain the relative abundances of the original sample, including low abundance transcripts. RNA can be amplified in vitro, in situ, or in vivo (See Eberwine U.S. Pat. No. 5,514,545).
It is also advantageous to include controls within the sample to assure that amplification and labeling procedures do not change the true distribution of nucleic acid molecules in a sample. For this purpose, a sample is spiked with an amount of a control nucleic acid molecule predetermined to be detectable upon hybridization to its complementary arrayed nucleic acid molecule and the composition of nucleic acid molecules includes reference nucleic acid molecules which specifically hybridize with the control arrayed nucleic acid molecules. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control arrayed nucleic acid molecules added to the sample.
Prior to hybridization, it may be desirable to fragment the sample nucleic acid molecules. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other sample nucleic acid molecules in the sample or noncomplementary nucleic acid molecules. Fragmentation can be performed by mechanical or chemical means.
Labeling
The sample nucleic acid molecules or probes may be labeled with one or more labeling moieties to allow for detection of hybridized arrayed/sample nucleic acid molecule complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical, or chemical means. The labeling moieties include radioisotopes, such as (32)P, (33)P or (35)S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like. Preferred fluorescent markers include Cy3 and Cy5 fluorophores (Amersham Pharmacia Biotech, Piscataway N.J.).
Hybridization
The nucleic acid molecule sequence of SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 or other CD163 encoding sequences in the art and fragments thereof can be used in various hybridization technologies for various purposes. Hybridization probes may be designed or derived from any mammalian CD163 sequence but may make use of those sequences disclosed in SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47. Such probes may be made from a highly specific region or from a conserved motif, and used in protocols to quantify CD163 message, allelic variants, or related sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from any mammalian CD163 sequence known in the art or from those sequences disclosed herein as SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 or from genomic sequences including promoters, enhancers, and introns of the mammalian gene. Hybridization or PCR probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of the labeled nucleotide. A vector containing the nucleic acid sequence may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleic acid molecules. These procedures may be conducted using commercially available kits such as those provided by Amersham Pharmacia Biotech.
The stringency of hybridization is determined by the G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane-based hybridizations, additions of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization can be performed at low stringency with buffers, such as 5× saline sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60° C., which permits the formation of a hybridization complex between nucleotide sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2×SSC with 0.1% SDS at either 45° C. (medium stringency) or 68° C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acid sequences are almost completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of other detergents such as Sarkosyl or Triton X-100 and a blocking agent such as salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.
Exemplary highly stringent hybridization conditions are as follows: hybridization at 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1×SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.
Hybridization specificity can be evaluated by comparing the hybridization of specificity-control nucleic acid molecules to specificity-control sample nucleic acid molecules that are added to a sample in a known amount. The specificity-control arrayed nucleic acid molecules may have one or more sequence mismatches compared with the corresponding arrayed nucleic acid molecules. In this manner, it is possible to determine whether only complementary arrayed nucleic acid molecules are hybridizing to the sample nucleic acid molecules or whether mismatched hybrid duplexes are forming is determined.
Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, nucleic acid molecules from one sample are hybridized to the molecules in a microarray format and signals detected after hybridization complex formation correlate to nucleic acid molecule levels in a sample. In the differential hybridization format, the differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, nucleic acid molecules from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled nucleic acid molecules is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Molecules in the microarray that are hybridized to substantially equal numbers of nucleic acid molecules derived from both biological samples give a distinct combined fluorescence (Shalon et al.; PCT publication WO95/35505). In a preferred embodiment, the labels are fluorescent markers with distinguishable emission spectra, such as Cy3 and Cy5 fluorophores.
After hybridization, the microarray is washed to remove nonhybridized nucleic acid molecules and complex formation between the hybridizable array elements and the nucleic acid molecules is detected. Methods for detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the nucleic acid molecules are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy.
In a differential hybridization experiment, nucleic acid molecules from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the nucleic acid molecules in two or more samples are obtained.
Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual arrayed-sample nucleic acid molecule complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
Polypeptide Based Assays
The present invention provides methods and reagents for detecting and quantifying CD163 polypeptides. These methods include analytical biochemical methods such as electrophoresis, mass spectroscopy, chromatographic methods and the like, or various immunological methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, affinity capture mass spectrometry, biological activity, and others described below and apparent to those of skill in the art upon review of this disclosure.
Immunoassays
The present invention also provides methods for detection of CD163 polypeptides employing one or more anti-CD163 antibody reagents (i.e., immunoassays). As used herein, an immunoassay is an assay that utilizes an antibody (as broadly defined herein and specifically includes fragments, chimeras and other binding agents) that specifically binds a CD163 polypeptide or epitope.
A number of well-established immunological binding assay formats suitable for the practice of the invention are known (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g., Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Ten, eds. (1991); Harlow and Lane, supra [e.g., Chapter 14], and Ausubel et al., supra, [e.g., Chapter 11]. Typically, immunological binding assays (or immunoassays) utilize a “capture agent” to specifically bind to and, often, immobilize the analyte to a solid phase. In one embodiment, the capture agent is a moiety that specifically binds to a CD163 polypeptide or subsequence, such as an anti-CD163 antibody.
Usually the CD163 gene product being assayed is detected directly or indirectly using a detectable label. The particular label or detectable group used in the assay is usually not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody or antibodies used in the assay. The label may be covalently attached to the capture agent (e.g., an anti-CD163 antibody), or may be attached to a third moiety, such as another antibody, that specifically binds to the CD163 polypeptide.
The present invention provides methods and reagents for competitive and noncompetitive immunoassays for detecting CD163 polypeptides. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case CD163) is directly measured. One such assay is a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the CD163 polypeptide. See, e.g., Maddox et al., 1983, J. Exp. Med., 158:1211 for background information. In one “sandwich” assay, the capture agent (e.g., an anti-CD163 antibody) is bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture any CD163 polypeptide present in the test sample. The CD163 polypeptide thus immobilized can then be labeled, e.g., by binding to a second anti-CD163 antibody bearing a label. Alternatively, the second CD163 antibody may lack a label, but be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody alternatively can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
In competitive assays, the amount of CD163 polypeptide present in the sample is measured indirectly by measuring the amount of an added (exogenous) CD163 polypeptide displaced (or competed away) from a capture agent (e.g., CD163 antibody) by the CD163 polypeptide present in the sample. A hapten inhibition assay is another example of a competitive assay. In this assay CD163 polypeptide is immobilized on a solid substrate. A known amount of CD163 antibody is added to the sample, and the sample is then contacted with the immobilized CD163 polypeptide. In this case, the amount of anti-CD163 antibody bound to the immobilized CD163 polypeptide is inversely proportional to the amount of CD163 polypeptide present in the sample. The amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. In this aspect, detection may be direct, where the antibody is labeled, or indirect where the label is bound to a molecule that specifically binds to the antibody as described above.
Other Antibody-Based Assay Formats
The invention also provides reagents and methods for detecting and quantifying the presence of CD163 polypeptide in the sample by using an immunoblot (Western blot) format. Another immunoassay is the so-called “lateral flow chromatography.” In a non-competitive version of lateral flow chromatography, a sample moves across a substrate by, e.g., capillary action, and encounters a mobile-labeled antibody that binds the analyte forming a conjugate. The conjugate then moves across the substrate and encounters an immobilized second antibody that binds the analyte. Thus, immobilized analyte is detected by detecting the labeled antibody. In a competitive version of lateral flow chromatography a labeled version of the analyte moves across the carrier and competes with unlabeled analyte for binding with the immobilized antibody. The greater the amount of the analyte in the sample, the less the binding by labeled analyte and, therefore, the weaker the signal. See, e.g., May et al., U.S. Pat. No. 5,622,871; and Rosenstein, U.S. Pat. No. 5,591,645.
Depending upon the assay, various components, including the antigen, target antibody, or anti-cathepsin S antibody, may be bound to a solid surface or support (i.e., a substrate, membrane, or filter paper). Many methods for immobilizing biomolecules to a variety of solid surfaces are known in the art. For instance, the solid surface may be a membrane (e.g., nitrocellulose), a microliter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass or plastic bead. The desired component may be covalently bound or noncovalently attached through nonspecific bonding.
A wide variety of organic and inorganic polymers, both natural and synthetic may be employed as the material for the solid surface. Illustrative polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other materials that may be employed, include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like. In addition, substances that form gels, such as proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used. Polymers that form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.
Mass Spectrometry
The mass of a molecule frequently can be used as an identifier of the molecule. Therefore, methods of mass spectrometry can be used to identify a protein analyte. Mass spectrometers can measure mass by determining the time required for an ionized analyte to travel down a flight tube and to be detected by an ion detector. One method of mass spectrometry for proteins is matrix-assisted laser desorption ionization mass spectrometry (“MALDI”). In MALDI the analyte is mixed with an energy absorbing matrix material that absorbs energy of the wavelength of a laser and placed on the surface of a probe. Upon striking the matrix with the laser, the analyte is desorbed from the probe surface, ionized, and detected by the ion detector. See, for example, Hillenkamp et al., U.S. Pat. No. 5,118,937.
Other methods of mass spectrometry for proteins are described in Hutchens and Yip, U.S. Pat. No. 5,719,060. In one such method referred to as Surfaces Enhanced for Affinity Capture (“SEAC”) a solid phase affinity reagent that binds the analyte specifically or non-specifically, such as an antibody or a metal ion, is used to separate the analyte from other materials in a sample. Then the captured analyte is desorbed from the solid phase by, e.g., laser energy, ionized, and detected by the detector.
Nucleic Acids of the Invention
The examples below disclose our discovery of several novel CD163 polynucleotides. The invention includes these novel CD163 polynucleotides. The present invention provides several isolated novel polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single and double-stranded, including splice variants thereof, which encode novel CD163 polypeptides. We report herein isolated novel polynucleotides which encode porcine, murine, human, canine, and african green monkey CD163 polypeptides and which comprise the sequences set forth in SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47.
It should be recognized that by disclosing SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 it provides one skilled in the art a multitude of methods of obtaining these sequences. By way of example, it would be possible to generate probes from the sequences disclosed in SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 and screen porcine, murine, human, canine, and african green monkey cDNA or genomic libraries and thereby obtain the entire sequence disclosed in SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or its genomic equivalent. Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989). Also by way of example, one skilled in the art would immediately recognize that given the sequence disclosed in SEQ ID NO: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 it is then possible to generate the appropriate primers for PCR amplification to obtain the entire sequence represented by these sequences. (See e.g., PCR Technology, H. A. Erlich, ed., Stockton Press, New York, 1989; PCR Protocols: A Guide to Methods and Applications, M. A. Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds., Academic Press, Inc., New York, 1990.)
DNA polynucleotides of the invention include cDNA, and DNA that has been chemically synthesized in whole or in part and is also intended to include allelic variants thereof. Allelic variants are modified forms of a wild type gene sequence, the modification resulting from recombination during chromosomal segregation, or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation).
DNA sequences encoding the novel CD163 polypeptides are set forth in SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47. The worker of skill in the art will readily appreciate that the DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ID NO: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence deducible from the sequences of SEQ ID NOs: 1, 5, 12, 13, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 according to Watson-Crick base pairing rules for DNA. Also contemplated by the invention are other polynucleotides encoding for the porcine, murine, and african green monkey CD163 polypeptides of SEQ ID NOS: 2, 14, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48 which differ in sequence from the polynucleotide of SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45, and 47 by virtue of the well-known degeneracy of the universal genetic code, as is well known in the art. The present invention, therefore, contemplates those other DNA and RNA molecules that, on expression, encode the polypeptides of SEQ ID NO: 2, 14, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48. Having identified the amino acid residue sequence encoded the porcine CD163 polypeptide, and with the knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein characterized simply by a change in a codon for a particular amino acid, are, therefore, within the scope of this invention.
A table of amino acids and their representative abbreviations, symbols and codons is set forth below in the following Table 4.










TABLE 4





Amino acid
Abbrev.
Symbol
Codon(s)







Alanine
Ala
A
GCA GCC GCG GCU


 


Cysteine
Cys
C
UGC UGU


 


Aspartic acid
Asp
D
GAC GAU


 


Glutamic acid
Glu
E
GAA GAG


 


Phenylalanine
Phe
F
UUC UUU


 


Glycine
Gly
G
GGA GGC GGG GGU


 


Histidine
His
H
CAC CAU


 


Isoleucine
Ile
I
AUA AUC AUU


 


Lysine
Lys
K
AAA AAG


 


Leucine
Leu
L
UUA UUG CUA CUC CUG CUU


 


Methionine
Met
M
AUG


 


Asparagine
Asn
N
AAC AAU


 


Proline
Pro
P
CCA CCC CCG CCU


 


Glutamine
Gln
Q
CAA CAG


 


Arginine
Arg
R
AGA AGG CGA CGC CGG CGU


 


Serine
Ser
S
AGC AGU UCA UCC UCG UCU


 


Threonine
Thr
T
ACA ACC ACG ACU


 


Valine
Val
V
GUA GUC GUG GUU


 


Tryptophan
Trp
W
UGG


 


Tyrosine
Tyr
Y
UAC UAU









As is well known in the art, codons constitute triplet sequences of nucleotides in mRNA and their corresponding cDNA molecules. Codons are characterized by the base uracil (U) when present in an mRNA molecule but are characterized by the base thymidine (T) when present in DNA. A simple change in a codon for the same amino acid residue within a polynucleotide will not change the sequence or structure of the encoded polypeptide. It is apparent that when a phrase stating that a particular 3 nucleotide sequence “encode(s)” any particular amino acid, the ordinarily skilled artisan would recognize that the table above provides a means of identifying the particular nucleotides at issue. By way of example, if a particular three-nucleotide sequence encodes theonine the table above discloses that the posible triplet sequences are ACA, ACG, ACC, and ACU (ACT if in DNA).
The invention includes therefore, an isolated polynucleotide comprising:

    (a) a susCD163v1 polynucleotide sequence set forth in SEQ ID NOs: 1 and 5;
    (b) a polynucleotide that encodes a polypeptide that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 2;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 2;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a susCD163v2 polynucleotide sequence set forth in SEQ ID NOs: 12 or 13;
    (b) a polynucleotide that encodes a polypeptide that has at least 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 14;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 14;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a susCD163v2 polynucleotide sequence set forth in SEQ ID NOs: 17 or 18;
    (b) a polynucleotide encoding a polypeptide of SEQ ID NO: 19;
    (d) a polynucleotide that is the complement of any of (a) or (b).


The invention also includes an isolated polynucleotide comprising:

    (a) a CD63v2 polynucleotide sequence set forth in SEQ ID NOs: 22 or 23;
    (b) a polynucleotide encoding a polypeptide of SEQ ID NO: 24;
    (c) a polynucleotide that is the complement of (a) or (b).


The invention also includes an isolated polynucleotide comprising:

    (a) a CD163v3 polynucleotide sequence set forth in SEQ ID NOs: 25 or 26;
    (b) a polynucleotide that encodes a polypeptide that has at least 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 27;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a CD163v2 polynucleotide sequence set forth in SEQ ID NOs: 30 or 31;
    (b) a polynucleotide that encodes a polypeptide that has at least 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 32;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 33;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 34;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 35;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 36;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 36;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 37;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 38
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 38;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 39;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 40;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 41;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 42;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 42;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 43;
    (b) a polynucleotide that encodes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 44;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 44;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 45;
    (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 46;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 46;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The invention also includes an isolated polynucleotide comprising:

    (a) a polynucleotide sequence set forth in SEQ ID NO: 47;
    (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 48;
    (c) a polynucleotide encoding a polypeptide of SEQ ID NO: 49;
    (d) a polynucleotide that is the complement of any of (a), (b), or (c).


The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related porcine CD163v1 polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and the polymerase chain reaction (PCR).
Knowledge of the sequence of any of the CD163 sequences disclosed herein also makes possible through use of Southern hybridization or polymerase chain reaction (PCR), the identification of genomic DNA sequences encoding CD163 regulatory sequences, such as promoters, operators, enhancers, repressors, and the like.
As noted in the section above entitled “Assays of the Invention” polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express CD163, or to measure levels of CD163 expression. Polynucleotides of the invention may also be the basis for diagnostic methods useful for determining the susceptibility of an animal to virus infection as described above.
The disclosure herein of the full-length polynucleotides encoding a CD163 polypeptide makes readily available to the worker of ordinary skill in the art fragments of the full length polynucleotide. The invention therefore provides unique fragments of the CD163 encoding polynucleotides comprising at least 15 through the length of the full-length sequence (including each and every integer value between) consecutive nucleotides of a polynucleotide encoding a CD163 disclosed herein. Because polynucleotides of the invention (including fragments) comprise sequences unique to the particular CD163-encoding polynucleotide sequence, they therefore would hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding the various CD163 polypeptides. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.
One or more unique fragment polynucleotides (or other CD163 polynucleotides as discussed above) can be included in kits that are used to detect the presence of a polynucleotide encoding for CD163, or used to detect variations in a polynucleotide sequence encoding for CD163. Also made available by the invention are anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding CD163. Full length and fragment anti-sense polynucleotides are provided. Fragment anti-sense molecules of the invention include (i) those that specifically recognize and hybridize to the CD163 variants disclosed herein (as determined by sequence comparison of DNA encoding CD163s to DNA encoding other known molecules). Identification of sequences unique to the novel CD163-encoding polynucleotides can be deduced through the use of any publicly available sequence database, and/or through the use of commercially available sequence comparison programs. The uniqueness of selected sequences in an entire genome can be further verified by hybridization analyses. After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Anti-sense polynucleotides are particularly relevant to regulating expression of CD163 by those cells expressing CD163 mRNA.
Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to CD163 expression control sequences or CD163 RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the porcine CD163 target nucleotide sequence in the cell and prevents transcription or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5′ end. Suppression of porcine CD163 expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases characterized by aberrant porcine CD163 expression or as a therapeutic modality.
As noted above in more detail, the nucleic acids of the invention include vectors comprising a polynucleotide of the invention. Such vectors are useful, e.g., for amplifying the polynucleotides in host cells to create useful quantities thereof. In other embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence. Such vectors are useful for recombinant production of polypeptides of the invention.
Also as noted above the invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated above, such host cells are useful for the production of virus and the production of vaccines.
The invention also provides isolated CD163 polypeptides encoded by a novel polynucleotide of the invention.
Polypeptides of the Invention
The examples disclose our discovery of several novel CD163 polypeptides. The invention includes these novel CD163 polypeptide which are set forth in SEQ ID NOs: 2, 14, 19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48.
The invention includes therefore, an isolated polynucleotide comprising a susCD163v1 polypeptide with the sequence set forth in SEQ ID NO: 2.
The invention also includes a polypeptide that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 2.
The invention includes therefore, an isolated polynucleotide comprising a susCD163v2 polypeptide with the sequence set forth in SEQ ID NO: 14.
The invention also includes a polypeptide that has at least 99%, identity and/or similarity to a sus CD163v2 polypeptide set forth in SEQ ID NO: 14.
The invention also includes a CD163v2 polypeptide having the sequence set forth in SEQ ID NO: 24.
The invention also includes a CD163v3 polypeptide having the sequence set forth in SEQ ID NO: 27.
The invention also includes a polypeptide having at least 96%, 97% 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 32.
The invention also includes a polypeptide that has at least 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 34.
The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 36.
The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 36.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 38.
The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 39.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 40.
The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 42.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 42.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 44.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 44.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 46.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 46.
The invention also includes a polypeptide having the sequence set forth in SEQ ID NO: 48.
The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 48.
Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of the novel CD163 polypeptides are embraced.
Overexpression in eukaryotic and prokaryotic hosts as described above facilitates the isolation of CD163 polypeptides. The invention therefore includes isolated CD163 polypeptides as set out in SEQ ID NOs: 2, 14, 19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48 and variants and conservative amino acid substitutions therein including labeled and tagged polypeptides.
The invention includes novel CD163 polypeptides that are “labeled”. The term “labeled” is used herein to refer to the conjugating or covalent bonding of any suitable detectable group, including enzymes (e.g., horseradish peroxidase, beta-glucuronidase, alkaline phosphatase, and beta-D-galactosidase), fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., 14C, 125I, 3H, 32P, and 35S) to the compound being labeled. Techniques for labeling various compounds, including proteins, peptides, and antibodies, are well known. See, e.g., Morrison, Methods in Enzymology 32b, 103 (1974); Syvanen et al., J. Biol. Chem. 284, 3762 (1973); Bolton and Hunter, Biochem. J. 133, 529 (1973). The termed labeled may also encompass a polypeptide which has covalently attached an amino acid tag as discussed below.
In addition, the novel CD163 polypeptides of the invention may be indirectly labeled. This involves the covalent addition of a moiety to the polypeptide and subsequent coupling of the added moiety to a label or labeled compound that exhibits specific binding to the added moiety. Possibilities for indirect labeling include biotinylation of the peptide followed by binding to avidin coupled to one of the above label groups. Another example would be incubating a radiolabeled antibody specific for a histidine tag with a CD163s polypeptide comprising a polyhistidine tag. The net effect is to bind the radioactive antibody to the polypeptide because of the considerable affinity of the antibody for the tag.
The invention also embraces variants (or analogs) of the novel CD163 protein. In one example, insertion variants are provided wherein one or more amino acid residues supplement a novel CD163 amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the novel CD163 protein amino acid sequence. Insertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels. Insertion variants include novel CD163 polypeptides wherein one or more amino acid residues are added to a CD163 acid sequence, or to a biologically active fragment thereof.
Insertional variants therefore can also include fusion proteins wherein the amino and/or carboxy termini of the novel CD163 polypeptide is fused to another polypeptide. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the influenza HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)], and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an alpha-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)]. In addition, the CD163 polypeptide can be tagged with enzymatic proteins such as peroxidase and alkaline phosphatase.
In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a novel CD163 polypeptide is removed. Deletions can be effected at one or both termini of the novel CD163 polypeptide, or with removal of one or more residues within the novel CD163 amino acid sequence. Deletion variants, therefore, include all fragments of the novel CD163 polypeptide.
CD163 polypeptides contain a transmembrane or membrane anchor region. It should be recognized that such transmembrane domains are useful when expressed in the context of a heterologous protein to aid in the targeting of the heterologous protein to membranes. It should also be recognized that it may be advantageous to delete some transmembrane domains to enhance the purification or solubility of the protein. Transmembrane deleted variants of CD163 and polynucleotides encoding them are of potential value as antiviral therapeutics. Such variants are specifically disclosed here as SEQ ID NOs: 37-40.
The present invention also includes variants of the aforementioned polypeptides, that is, polypeptides that vary from the reference sequence by conservative amino acid substitutions,
Exemplary conservative substitutions are set out in Tables 1, 2 and 3 in the section above entitled “Definitions”.
In those situations where it is preferable to partially or completely isolate the novel CD163 polypeptides, purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange), and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification.
Purification of novel CD163 polypeptides can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (CD163/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i.e., a monoclonal antibody specifically recognizing CD163). For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen Registered TM nickel columns) can be used for purification of CD163/polyHis. (See for example, Ausubel et al., eds., Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York [1993]).
Even if the novel CD163 polypeptide is prepared without a label or tag to facilitate purification, the novel CD163 of the invention may be purified by immunoaffinity chromatography. To accomplish this, antibodies specific for CD163 polypeptides must be prepared by means well known in the art.
Antibodies generated against the novel CD163 polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues, or cells to an animal, preferably a non-human, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).
Where the novel CD163 polypeptides are prepared without a tag attached, and no antibodies are available, other well-known procedures for purification can be used. Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific). In some cases, two or more of these techniques may be combined to achieve increased purity.
It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.
Antibodies
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for novel CD163 or fragments thereof.
The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind CD163s polypeptides exclusively (i.e., able to distinguish CD163s polypeptides from other known polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between the novel CD163 and such polypeptides). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the CD163s polypeptides of the invention are also contemplated, provided that the antibodies are, first and foremost, specific for novel CD163 polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art. Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
Antibodies of the invention are useful for, diagnostic purposes to detect or quantitate CD163s, as well as purification of CD163s. Kits comprising an antibody of the invention for any of the purposes described herein are also contemplated. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.
The present invention is further illustrated, but not limited, by the following examples.
Example 1
Transient Transfection with Porcine CD163 Confers Permissivity to PRRS Virus Infection to a Non-Permissive Cell Line
Total mRNA from primary porcine alveolar macrophage cells was used to construct a cDNA library in the plasmid pCMV-Sport6.1 (Invitrogen), with the cDNA cloned between the EcoRV and NotI sites. A member of this library, when isolated and transiently transfected into the BHK-21 (baby hamster kidney) cell line, conferred a PRRS-permissive phenotype. Cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in a 5% CO2 atmosphere at 37° C. Cell cultures were transiently transfected using 10.0 uL of Lipofectamine 2000 (Invitrogen) and 2.0 ug of plasmid. A duplicate monolayer was transfected with negative control plasmid pPAMB. This plasmid is pCMV-Sport6.1 lacking an insert. Transfection efficiency was monitored with a plasmid expressing green fluorescent protein (GFP). Approximately 24 hours post-transfection, monolayers were infected with either North American (isolate P129) or European (isolate 96V198) genotypes of PRRS virus. For detection of PRRS replication, the monolayers were fixed using 80% acetone approximately 24 hours post-infection and incubated for approximately 1 hour with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc.). This monoclonal antibody is specific for PRRS viral nucleocapsid expressed from open reading frame 7. A Nikon TE 300 inverted fluorescent microscope with a 10× objective was used to photograph a monolayer containing FITC positive cells and a negative control monolayer.
It was confirmed that transfected cells became permissive to both the North American (isolate P129) and European (isolate 96V198) genotypes of PRRSV. Expression of viral genes could be detected in many of the transfected BHK cells, and progeny virus was readily detectable in the supernatant. Control transfections using vector without insert or irrelevant plasmids did not confer permissivity.
Sequencing of the insert in the functional plasmid, using the Big Dye Terminator Version 1.0 Sequence Reaction kit (Applied Biosystems, Foster City, Calif.) and the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems), revealed a gene that was highly homologous to the published porcine CD163 gene cDNA (Genbank accession number AJ311716). The cDNA we identified contained additional 5′ and 3′ untranslated regions relative to AJ311716, and the open reading frame differed in three ways: (1) a 738 by internal deletion near the 5′ end, (2) a 15 by extension of the 5′ end to an upstream ATG codon, and (3) sixteen nucleotide changes predicted to cause 10 amino acid changes. Nucleotide sequence identity between the sequences was 99.4%. Alignments of the newly discovered porcine CD163 sequence with the previously reported sequence AJ311716 are shown in FIGS. 1 and 2. The novel porcine CD163 variant was designated “susCD163v1”.












SEQUENCE

ID NO







gtaataatac aagaagattt aaatgggcat aaaaccttgg aatggacaaa ctcagaatgg
  60
SEQ ID


 


tgctacatga aaactctgga tctgcagacc tgaaactgag agtggtagat ggagtcactg
 120
NO: 5


 


aatgttcagg aagattggaa gtgaaattcc aaggagaatg gggaacaatc tgtgatgatg
 180



 


gctgggatag tgatgatgcc gctgtggcat gtaagcaact gggatgtcca actgctgtca
 240



 


ctgccattgg tcgagttaac gccagtgagg gaactggaca catttggctt gacagtgttt
 300



 


cttgccatgg acacgagtct gctctctggc agtgtagaca ccatgaatgg ggaaagcatt
 360



 


attgcaatca taatgaagat gctggtgtga catgttctga tggatcagat ctggaactga
 420



 


gacttaaagg tggaggcagc cactgtgctg ggacagtgga ggtggaaatt cagaaactgg
 480



 


taggaaaagt gtgtgataga agctggggac tgaaagaagc tgatgtggtt tgcaggcagc
 540



 


tgggatgtgg atctgcactc aaaacatcat atcaagttta ttccaaaacc aaggcaacaa
 600



 


acacatggct gtttgtaagc agctgtaatg gaaatgaaac ttctctttgg gactgcaaga
 660



 


attggcagtg gggtggactt agttgtgatc actatgacga agccaaaatt acctgctcag
 720



 


cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac
 780



 


aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg
 840



 


tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg
 900



 


gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc
 960



 


tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg
1020



 


gcgtagtctg ctcaagatac acacaaatcc gcttggtgaa tggcaagacc ccatgtgaag
1080



 


gaagagtgga gctcaacatt cttgggtcct gggggtccct ctgcaactct cactgggaca
1140



 


tggaagatgc ccatgtttta tgccagcagc ttaaatgtgg agttgccctt tctatcccgg
1200



 


gaggagcacc ttttgggaaa ggaagtgagc aggtctggag gcacatgttt cactgcactg
1260



 


ggactgagaa gcacatggga gattgttccg tcactgctct gggcgcatca ctctgttctt
1320



 


cagggcaagt ggcctctgta atctgctcag ggaaccagag tcagacacta tccccgtgca
1380



 


attcatcatc ctcggaccca tcaagctcta ttatttcaga agaaaatggt gttgcctgca
1440



 


tagggagtgg tcaacttcgc ctggtcgatg gaggtggtcg ttgtgctggg agagtagagg
1500



 


tctatcatga gggctcctgg ggcaccatct gtgatgacag ctgggacctg aatgatgccc
1560



 


atgtggtgtg caaacagctg agctgtggat gggccattaa tgccactggt tctgctcatt
1620



 


ttggggaagg aacagggccc atttggctgg atgagataaa ctgtaatgga aaagaatctc
1680



 


atatttggca atgccactca catggttggg ggcggcacaa ttgcaggcat aaggaggatg
1740



 


caggagtcat ctgctcggag ttcatgtctc tcagactgat cagtgaaaac agcagagaga
1800



 


cctgtgcagg gcgcctggaa gttttttaca acggagcttg gggcagcgtt ggcaagaata
1860



 


gcatgtctcc agccacagtg ggggtggtat gcaggcagct gggctgtgca gacagagggg
1920



 


acatcagccc tgcatcttca gacaagacag tgtccaggca catgtgggtg gacaatgttc
1980



 


agtgtcctaa aggacctgac accctatggc agtgcccatc atctccatgg aagaagagac
2040



 


tggccagccc ctcagaggag acatggatca catgtgccaa caaaataaga cttcaagaag
2100



 


gaaacactaa ttgttctgga cgtgtggaga tctggtacgg aggttcctgg ggcactgtgt
2160



 


gtgacgactc ctgggacctt gaagatgctc aggtggtgtg ccgacagctg ggctgtggct
2220



 


cagctttgga ggcaggaaaa gaggccgcat ttggccaggg gactgggccc atatggctca
2280



 


atgaagtgaa gtgcaagggg aatgaaacct ccttgtggga ttgtcctgcc agatcctggg
2340



 


gccacagtga ctgtggacac aaggaggatg ctgctgtgac gtgttcagaa attgcaaaga
2400



 


gccgagaatc cctacatgcc acaggtcgct catcttttgt tgcacttgca atctttgggg
2460



 


tcattctgtt ggcctgtctc atcgcattcc tcatttggac tcagaagcga agacagaggc
2520



 


agcggctctc agttttctca ggaggagaga attctgtcca tcaaattcaa taccgggaga
2580



 


tgaattcttg cctgaaagca gatgaaacgg atatgctaaa tccctcagga gaccactctg
2640



 


aagtacaatg aaaaggaaaa tgggaattat aacctggtga gttcagcctt taagatacct
2700



 


tgatgaagac ctggactatt gaatgagcaa gaatctgcct cttacactga agattacaat
2760



 


acagtcctct gtctcctggt attccaaaga ctgctgttga atttctaaaa aatagattgg
2820



 


tgaatgtgac tactcaaagt tgtatgtaag actttcaagg gcattaaata aaaaagaata
2880



 


ttgctgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2930



 









   1
M  D  K  L  R  M  V  L  H  E  N  S  G  S  A  D  L  K  L  R
SEQ ID


   1
atggacaaactcagaatggtgctacatgaaaactctggatctgcagacctgaaactgaga
NO: 1


 


  21
V  V  D  C  V  T  E  C  S  G  R  L  E  V  K  F  Q  G  E  W
and 2


  61
gtggtagatggagtcactgaatgttcaggaagattggaagtgaaattccaaggagaatgg



 


  41
G  T  I  C  D  D  G  W  D  S  D  D  A  A  V  A  C  K  Q  L



 121
ggaacaatctgtgatgatggctgggatagtgatgatgccgctgtggcatgtaagcaactg



 


  61
G  C  P  T  A  V  T  A  I  G  R  V  N  A  S  E  G  T  G  H



 181
ggatgtccaactgctgtcactgccattggtcgagttaacgccagtgagggaactggacac



 


  81
I  W  L  D  S  V  S  C  H  G  H  E  S  A  L  W  Q  C  R  H 



 241
atttggcttgacagtgtttcttgccatggacacgagtctgctctctggcagtgtagacac



 


 101
H  E  W  G  K  H  Y  C  N  H  N  E  D  A  G  V  T  C  S  D



 301
catgaatggggaaagcattattgcaatcataatgaagatgctggtgtgacatgttctgat



 


 121
G  S  D  L  E  L  R  L  K  G  G  G  S  H  C  A  G  T  V  E



 361
ggatcagatctggaactgagacttaaaggtggaggcagccactgtgctgggacagtggag



 


 141
V  E  I  Q  K  L  V  G  K  V  C  D  N  S  W  G  L  K  E  A



 421
gtggaaattcagaaactggtaggaaaagtgtgtgatagaagctggggactgaaagaagct



 


 161
D  V  V  C  R  Q  L  G  C  G  S  A  L  K  T  S  Y  Q  V  Y



 481
gatgtggtttgcaggcagctgggatgtggatctgcactcaaaacatcatatcaagtttat



 


 181
S  K  T  K  A  T  N  T  W  L  F  V  S  S  C  N  G  N  E  T



 541
tccaaaaccaaggcaacaaacacatggctgtttgtaagcagctgtaatggaaatgaaact



 


 201
S  L  W  D  C  K  N  W  Q  W  G  G  L  S  C  D  H  Y  D  E



 601
tctctttgggactgcaagaattggcagtggggtggacttagttgtgatcactatgacgaa



 


 221
A  K  I  T  C  S  A  H  R  K  P  R  L  V  G  G  D  I  P  C



 661
gccaaaattacctgctcagcccacaggaaacccaggctggttggaggggacattccctgc



 


 241
S  G  R  V  E  V  Q  H  G  D  T  W  G  T  V  C  D  S  D  F



 721
tctggtcgtgttgaagtacaacatggagacacgtggggcaccgtctgtgattctgacttc



 


 261
S  L  E  A  A  S  V  L  C  R  E  L  Q  C  G  T  V  V  S  L



 781
tctctggaggcggccagcgtgctgtgcagggaactacagtgcggcactgtggtttccctc



 


 281
L  G  G  A  H  F  G  E  G  S  G  Q  I  W  A  E  E  F  Q  C



 841
ctggggggagctcactttggagaaggaagtggacagatctgggctgaagaattccagtgt



 


 301
E  G  H  E  S  H  L  S  L  C  P  V  A  P  R  P  D  G  T  C



 901
gaggggcacgagtoccacctttcactctgcccagtagcaccccgccctgacgggacatgt



 


 321
S  H  S  R  D  V  G  V  V  C  S  R  Y  T  Q  I  R  L  V  N



 961
agccacagcagggacgtcggcgtagtctgctcaagatacacacaaatccgcttggtgaat



 


 341
G  K  T  S  C  E  G  R  V  E  L  N  I  L  G  S  W  G  S  L



1021
ggcaagaccccatgtgaaggaagagtggagctcaacattcttgggtcctgggggtccctc



 


 361
C  N  S  H  W  D  M  E  D  A  H  V  L  C  Q  Q  L  K  C  G



1081
tgcaactctcactgggacatggaagatgcccatgttttatgccagcagcttaaatgtgga



 


 381
V  A  L  S  I  P  G  G  A  P  F  G  K  G  S  E  Q  V  W  R



1141
gttgccctttctatcccgggaggagcaccttttgggaaaggaagtgagcaggtctggagg



 


 401
H  M  F  H  C  T  G  T  E  K  H  M  G  D  C  S  V  T  A  L



1201
cacatgtttcactgcactgggactgagaagcacatgggagattgttccgtcactgctctg



 


 421
G  A  S  L  C  S  S  G  Q  V  A  S  V  I  C  S  G  N  Q  S



1261
ggcgcatcactctgttcttcagggcaagtggcctctgtaatctgctcagggaaccagagt



 


 441
Q  T  L  S  P  C  N  S  S  S  S  D  P  S  S  S  I  I  S  E



1321
cagacactatccccgtgcaattcatcatcctcggacccatcaagctctattatttcagaa



 


 461
E  N  G  V  A  C  I  G  S  G  Q  L  R  L  V  D  G  G  G  R



1381
gaaaatggtgttgcctgcatagggagtggtcaacttcgcctggtcgatggaggtggtcgt



 


 481
C  A  G  R  V  E  V  Y  H  E  G  S  W  G  T  I  C  D  D  S



1441
tgtgctgggagagtagaggtctatcatgagggctcctggggcaccatctgtgatgacagc



 


 501
W  D  L  N  D  A  H  V  V  C  K  Q  L  S  C  G  W  A  I  N



1501
tgggacctgaatgatgcccatgtggtgtgcaaacagctgagctgtggatgggccattaat



 


 521
A  T  G  S  A  H  F  G  E  G  T  G  P  I  W  L  D  E  I  N



1561
gccactggttctgctcattttggggaaggaacagggcccatttggctggatgagataaac



 


 541
C  N  G  K  E  S  H  I  W  Q  C  H  S  H  G  W  G  R  H  N



1621
tgtaatggaaaagaatctcatatttggcaatgccactcacatggttgggggcggcacaat



 


 561
C  R  H  K  E  D  A  G  V  I  C  S  E  F  M  S  L  R  L  I



1681
tgcaggcataaggaggatgcaggagtcatctgctcggagttcatgtctctcagactgatc



 


 581
S  E  N  S  R  E  T  C  A  G  R  L  E  V  F  Y  N  G  A  W



1741
agtgaaaacagcagagagacctgtgcagggcgcctggaagttttttacaacggagcttgg



 


 601
G  S  V  G  K  N  S  M  S  P  A  T  V  G  V  V  C  R  Q  L



1801
ggcagcgttggcaagaatagcatgtctccagccacagtgggggtggtatgcaggcagctg



 


 621
G  C  A  D  R  G  D  I  S  P  A  S  S  D  K  T  V  S  R  H



1861
ggctgtgcagacagaggggacatcagccctgcatcttcagacaagacagtgtccaggcac



 


 641
M  W  V  D  N  V  Q  C  P  K  G  P  D  T  L  W  Q  C  P  S



1921
atgtgggtggacaatgttcagtgtcctaaaggacctgacaccctatggcagtgcccatca



 


 661
S  P  W  K  K  R  L  A  S  P  S  E  E  T  W  I  T  C  A  N



1981
tctccatggaagaagagactggccagcccctcagaggagacatggatcacatgtgccaac



 


 681
K  I  R  L  Q  E  G  N  T  N  C  S  G  R  V  E  I  W  Y  G



2041
aaaataagacttcaagaaggaaacactaattgttctggacgtgtggagatctggtacgga



 


 701
G  S  W  G  T  V  C  D  D  S  W  D  L  E  D  A  Q  V  V  C



2101
ggttcctggggcactgtgtgtgacgactcctgggaccttgaagatgctcaggtggtgtgc



 


 721
R  Q  L  G  C  G  S  A  L  E  A  G  K  E  A  A  F  G  Q  G



2161
cgacagctgggctgtggctcagctttggaggcaggaaaagaggccgcatttggccagggg



 


 741
T  G  P  I  W  L  N  E  V  K  C  K  G  N  E  T  S  L  W  D



2221
actgggcccatatggctcaatgaagtgaagtgcaaggggaatgaaacctccttgtgggat



 


 761
C  P  A  R  S  W  G  H  S  D  C  G  H  K  E  D  A  A  V  T



2281
tgtcctgccagatcctggggccacagtgactgtggacacaaggaggatgctgctgtgacg



 


 781
C  S  E  I  A  K  S  R  E  S  L  H  A  T  G  R  S  S  F  V



2341
tgttcagaaattgcaaagagccgagaatccctacatgccacaggtcgctcatcttttgtt



 


 801
A  L  A  I  F  G  V  I  L  L  A  C  L  I  A  F  L  I  W  T



2401
gcacttgcaatctttggggtcattctgttggcctgtctcatcgcattcctcatttggact



 


 821
Q  K  R  R  Q  R  Q  R  L  S  V  F  S  G  G  E  N  S  V  H



2461
cagaagcgaagacagaggcagcggctctcagttttctcaggaggagagaattctgtccat



 


 841
Q  I  Q  Y  R  E  M  N  S  C  L  K  A  D  E  T  D  M  L  N



2521
caaattcaataccgggagatgaattcttgcctgaaagcagatgaaacggatatgctaaat



 


 861
P  S  G  D  H  S  E  V  Q



2581
ccctcaggagaccactctgaagtacaa



 


   1
MDKLRMVLHE NSGSADLKLR VVDGVTECSG RLEVKFQGEW GTICDDGWDS
SEQ ID


 


  51
DDAAVACKQL GCPTAVTAIG RVNASEGTGH IWLDSVSCHG HESALWQCRH
NO: 2


 


 101
HEWGKHYCNH NEDAGVTCSD GSDLELRLKG GGSHCAGTVE VEIQKLVGKV



 


 151
CDRSWGLKEA DVVCRQLGCG SALKTSYQVY SKTKATNTWL FVSSCNGNET



 


 201
SLWDCKNWQW GGLSCDHYDE AKITCSAHRK PRLVGGDIPC SGRVEVQHGD



 


 251
TWGTVCDSDF SLEAASVLCR ELQCGTVVSL LGGAHFGEGS GQIWAEEFQC



 


 301
EGHESHLSLC PVAPRPDGTC SHSRDVGVVC SRYTQIRLVN GKTPCEGRVE



 


 351
LNILGSWGSL CNSHWDMEDA HVLCQQLKCG VALSIPGGAP FGKGSEQVWR



 


 401
HMFHCTGTEK HMGDCSVTAL GASLCSSGQV ASVICSGNQS QTLSPCNSSS



 


 451
SDPSSSIISE ENGVACIGSG QLRLVDGGGR CAGRVEVYHE GSWGTICDDS



 


 501
WDLNDAHVVC KQLSCGWAIN ATGSAHFGEG TGPIWLDEIN CNGKESHIWQ



 


 551
CHSHGWGRHN CRHKEDAGVI CSEFMSLRLI SENSRETCAG RLEVFYNGAW



 


 601
GSVGKNSMSP ATVGVVCRQL GCADRGDISP ASSDKTVSRH MWVDNVQCPK



 


 651
GPDTLWQCPS SPWKKRLASP SEETWITCAN KIRLQEGNTN CSGRVEIWYG



 


 701
GSWGTVCDDS WDLEDAQVVC RQLGCGSALE AGKEAAFGQG TGPIWLNEVK



 


 751
CKGNETSLWD CPARSWGHSD CGHKEDAAVT CSEIAKSRES LHATGASSFV



 


 801
ALAIFGVILL ACLIAFLIWT QKRRQRQRLS VFSGGENSVH QIQYREMNSC



 


 851
LKADETDMLN PSGDHSEVQ









Example 2
Construction of Plasmid pCMVsusCD163v1
Construction of the plasmid pCMVsusCD163v1 was performed as follows. The functional clone identified in the primary porcine macrophage cDNA library as conferring PRRSV permissivity served as template for PCR amplification of the CD163 insert, including the 5′ and 3′ untranslated regions, using the primers 5′DS-CD163 (SEQ ID NO:6) (5′-CGGAATTCCGCGGATGTAATAATACAAGAAGA-3′) and 3′CD163 (SEQ ID NO:7) (5′CCGCTCGAGTAGTCCAGGTCTTCATCAAGGTATCTT-3′). Primer 5′DS-CD163 incorporates a SacII restriction site at the 5′ end of the CD163 insert, while primer 3′CD163 incorporates an XhoI restriction site at the 3′ end of the insert (underlined). Reactions containing 190 ng of plasmid template were amplified using Platinum Pfx DNA polymerase (Invitrogen cat #11708-013) following the manufacture's instructions. Reactions were heated to 94° for 2 minutes then cycled 35 times through 94° for 20 seconds, 55° for 30 seconds, and 68° for 3.5 minutes followed by a terminal extension at 72° for 7 minutes. The resulting PCR products were purified using the Qiaquick PCR purification kit (Qiagen cat #28104), digested with restriction enzymes SacII and XhoI, and the resulting fragments were gel purified using the Qiaquick Gel Extraction kit (Qiagen cat #28704). The CD163 PCR fragment was then ligated into the plasmid pCMV-Script (Stratagene cat #212220) prepared to accept the digested PCR fragment by digestion with SacII and XhoI followed by gel purification as described above. The ligated material was transformed into E. coli strain DH5α and recombinants were selected by growth in 50 μg/ml kanamycin and identified by restriction analysis. The resulting plasmid, “pCMVsusCD163v1”, contains the internally deleted porcine CD163 insert described in Example 1 under the transcriptional control of the eukaryotic CMV promoter and the neomycin/kanamycin resistance gene under the control of both eukaryotic and prokaryotic promoters.
Example 3
Construction of the pRSV-Script Expression Vector and pRSVsusCD163v1
The plasmid pRc/RSV (Invitrogen) was used as a template for PCR amplification of the RSV promoter. RSV promoter sequence was contained within nucleotides 209 through 604 of pRc/RSV. Forward primer PCIRSVLTR (SEQ ID NO:8) (5′-ACACTCGACATGTCGATGTACGGGCCAGATATACGCGT-3′) and reverse primer VSRRTLSAC (SEQ ID NO: 9) (5′TTCCTTACAGAGCTCGAGGTGCACACCAATGTGGTGAA-3′) were synthesized. Restriction endonuclease Pci I and Sac I recognition sites (underlined) were incorporated into the 5′ and 3′ primers, respectively, for future cloning. PCR was performed using the HotMaster Taq DNA Polymerase kit (Eppendorf) following the manufacturer's instructions. The reactions contained 0.9 ng of pRc/RSV plasmid template and 0.3 μM of each primer described above. The reactions were heated to 94° for 2 minutes then cycled 30 times through 94° for 20 seconds, 52° for 10 seconds, and 65° for 1 minute. The resulting PCR fragment was digested with restriction enzymes Pci I and Sac I, gel purified, and cloned into the plasmid pCMV-Script (Stratagene) that had been similarly digested to remove the CMV promoter sequence. The final construct placed the RSV promoter immediately upstream of the multiple cloning site, and was named “pRSV-Script”.
The susCD163v1 insert was cloned behind the RSV promoter as follows. The susCD163v1 sequence was excised from plasmid pCMVsusCD163v1 by restriction digestion (Kpn I and Sac II) and gel purified. This fragment was ligated into pRSV-Script which had also been digested with the same enzymes and gel purified. The ligation mixture was transformed into DH5α E. coli and transformants selected using kanamycin at 50 μg/ml. The clone contained the correct insert was designed “pRSVsusCD163v1”.
Example 4
Cloning and Characterization of a Longer Variant of Porcine CD163 cDNA
Based on the porcine CD163v1 sequence, a forward primer 5′CD163NotIlong (SEQ ID NO:10) (5′CGGTCCGGAGCGGCCGCGATGTAATAATACAAGAAGATTTAAATGG-3′) and a reverse primer 3′CD163 KpnI (SEQ ID NO:11) (5′CGGTTGGTACCCAGCAATATTCTTTTTTATTTAATGCC-3′) were designed using the Lasergene PrimerSelect program (DNASTAR Inc., Madison Wis.) for amplification of a full-length porcine CD163 gene. Restriction endonuclease sites for Not I and Kpn I (underlined) were included in 5′ and 3′ primers, respectively, to allow for convenient cloning. Total cellular RNA was prepared from primary alveolar macrophages (PAM) harvested from lung lavages of healthy pigs. RNA preparation was done using the RNeasy mini kit (Qiagen, Valencia, Calif.). RT-PCR reactions were prepared using the SuperScript one-step RT-PCR for Long Templates kit (Invitrogen, Carlsbad, Calif.) and RT-PCR parameters were set as follows: 50° C. for 30 min, 94° C. for 2 min, (94° C. 30 sec, 55° C. 30 sec and 68° C. 4 min) for 35 cycles, 72° C. for 10 min. PCR products were analyzed on 0.8% SeaKem GTG agarose gels. RT-PCR products of various sizes were cut from agarose gels and DNA was extracted using the GeneClean kit (QBiogene). These RT-PCR products were cloned into the pCR2.1-TOPO cloning vector (Invitrogen). Clones were analyzed by restriction enzyme digestion for the presence of an insert. Colonies containing inserts were sequenced using Big Dye Terminator Version 1.0 Sequence Reaction kit (Applied Biosystems, Foster City, Calif.) and Applied Biosystems 3730 DNA Analyzer (Applied Biosystems) to confirm sequence authenticity. Sequences were edited and assembled using the Lasergene EditSeq and SeqMan programs (DNASTAR Inc., Madison Wis.). One plasmid with a large insert was designated “pCRsusCD163v2” (pCR2.1 containing porcine CD163 variant 2 which we have designated SEQ ID NO:12). The coding sequence contained within SEQ ID NO:12 is reproduced below and is designated SEQ ID NO:13. Sequence analysis showed that this porcine CD163 encodes an amino acid sequence of 1115 amino acids which we have designated SEQ ID NO:14. When compared to the porcine CD163 sequence in GenBank (Accession No. AJ311716), our CD163v2 sequence is 98.9% identical at the amino acid level. CD163v2 also has an additional 5 amino acid residues at the extreme 5′ end, extending the open reading frame to an in-frame upstream ATG initiation codon (as in the porcine CD163v1 sequence described in example 1). Porcine CD163 is 84.3% identical to human CD163 (GenBank Accession No. Z22968), and 73.7% identical to mouse CD163 (GenBank Accession No. AF274883) at the amino acid level. The predicted signal sequence and transmembrane region of SEQ ID NO:14 are indicated by underlining and bolding respectively. To determine whether other CD163 sequences contain similar sequence features is easily determined by inspection of the sequence.












SEQUENCE

ID NO







gtaataatac aagaagattt aaatggcata aaaccttgga atggacaaac tcagaatggt
  60
SEQ ID


 


gctacatgaa aactctggat ctgcagactt tagaagatgt tctgcccatt taagttcctt
 120
NO: 12


 


cacttttgct gtagtcgctg ttctcagtgc ctgcttggtc actagttctc ttggaggaaa
 180



 


agacaaggag ctgaggctaa cgggtggtga aaacaagtgc tctggaagag tggaggtgaa
 240



 


agtgcaggag gagtggggaa ctgtgtgtaa taatggctgg gacatggatg tggtctctgt
 300



 


tgtttgtagg cagctgggat gtccaactgc tatcaaagcc actggatggg ctaattttag
 360



 


tgcaggttct ggacgcattt ggatggatca tgtttcttgt cgagggaatg agtcagctct
 420



 


ctgggactgc aaacatgatg gatggggaaa gcataactgt actcaccaac aggatgctgg
 480



 


agtaacctgc tcagatggat ctgatttaga gatggggctg gtgaatggag gaaaccggtg
 540



 


cttaggaaga atagaagtca aatttcaagg acggtgggga acagtgtgtg atgataactt
 600



 


caacataaat catgcttctg tggtttgtaa acaacttgaa tgtggaagtg ctgtcagttt
 660



 


ctctggttca gctaattttg gagaaggttc tggaccaatc tggtttgatg atcttgtatg
 720



 


caatggaaat gagtcagctc tctggaactg caaacatgaa ggatggggaa agcacaattg
 780



 


cgatcatgct gaggatgctg gagtgatttg cttaaatgga gcagacctga aactgagagt
 840



 


ggtagatgga gtcactgaat gttcaggaag attggaagtg aaattccaag gagaatgggg
 900



 


aacaatctgt gatgatggct gggatagtga tgatgccgct gtggcatgta agcaactggg
 960



 


atgtccaact gctgtcactg ccattggtcg agttaacgcc agtgagggaa ctggacacat
1020



 


ttggcttgac agtgtttctt gccatggaca cgagtctgct ctctggcagt gtagacacca
1080



 


tgaatgggga aagcattatt gcaatcatga tgaagatgct ggtgtgacat gttctgatgg
1140



 


atcagatctg gaactgagac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt
1200



 


ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga
1260



 


tgtggtttgc aggcagctgg gatgtggatc tgcactcaaa acatcatatc aagtttattc
1320



 


caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc
1380



 


tctttgggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaagc
1440



 


caaaattacc tgctcagccc acaggaaacc caggctggtt ggaggggaca ttccctgctc
1500



 


tggtcgtgtt gaagtacaac atggagacac gtggggcacc gtctgtgatt ctgacttctc
1560



 


tctggaggcg gccagcgtgc tgtgcaggga actacagtgc ggcactgtgg tttccctcct
1620



 


ggggggagct cactttggag aaggaagtgg acagatctgg gctgaagaat tccagtgtga
1680



 


ggggcacgag tcccaccttt cactctgccc agtagcaccc cgccctgacg ggacatgtag
1740



 


ccacagcagg gacgtcggcg tagtctgctc aagatacaca caaatccgct tggtgaatgg
1800



 


caagacccca tgtgaaggaa gagtggagct caacattctt gggtcctggg ggtccctctg
1860



 


caactctcac tgggacatgg aagatgccca tgttttatgc cagcagctta aatgtggagt
1920



 


tgccctttct atcccgggag gagcaccttt tgggaaagga agtgagcagg tctggaggca
1980



 


catgtttcac tgcactggga ctgagaagca catgggagat tgttccgtca ctgctotggg
2040



 


cgcatcactc tgttcttcag ggcaagtggc ctctgtaatc tgctcaggga accagagtca
2100



 


gacactatct ccgtgcaatt catcatcctc ggacccatca agctctatta tttcagaaga
2160



 


aaatggtgtt gcctgcatag ggagtggtca acttcgcctg gtcgatggag gtggtcgttg
2220



 


tgctgggaga gtagaggtct atcatgaggg ctcctggggc accatctgtg atgacagctg
2280



 


ggacctgaat gatgcccatg tggtgtgcaa acagctgagc tgtggatggg ccattaatgc
2340



 


cactggttct gctcattttg gggaaggaac agggcccatt tggctggatg agataaactg
2400



 


taatggaaaa gaatctcata tttggcaatg ccactcacat ggttgggggc ggcacaattg
2460



 


caggcataag gaggatgcag gagtcatctg ctcagagttc atgtctctga gactgatcag
2520



 


tgaaaacagc agagagacct gtgcagggcg cctggaagtt ttttacaacg gagcttgggg
2580



 


cagcgttggc aggaatagca tgtctccagc cacagtgggg gtggtatgca ggcagctggg
2640



 


ctgtgcagac agaggggaca tcagccctgc atcttcagac aagacagtgt ccaggcacat
2700



 


gtgggtggac aatgttcagt gtcctaaagg acctgacaca ctatggcagt gcccatcatc
2760



 


tccatggaag aagagactgg ccagcccctc agaggagaca tggatcacat gtgccaacaa
2820



 


aataagactt caagaaggaa acactaattg ttctggacgt gtggagatct ggtacggagg
2880



 


ttcctggggc actgtgtgtg acgactcctg ggaccttgaa gatgctcagg tggtgtgccg
2940



 


acagctgggc tgtggctcag ctttggaggc aggaaaagag gccgcatttg gccaggggac
3000



 


tgggcccata tggctcaatg aagtgaagtg caaggggaat gaaacctcct tgtgggattg
3060



 


tcctgccaga tcctggggcc acagtgactg tggacacaag gaggatgctg ctgtgacgtg
3120



 


ctcagaaatt gcaaagagcc gagaatccct acatgccaca ggtcgctcat cttttgttgc
3180



 


acttgcaatc tttggggtca ttctgttggc ctgtctcatc gcattcctca tttggactca
3240



 


gaagcgaaga cagaggcagc ggctctcagt tttctcagga ggagagaatt ctgtccatca
3300



 


aattcaatac cgggagatga attcttgcct gaaagcagat gaaacggata tgctaaatcc
3360



 


ctcaggagac cactctgaag tacaatgaaa aggaaaatgg gaattataac ctggtgagtt
3420



 


cagcctttaa gataccttga tgaagacctg gactattgaa tgagcaagaa tctgcctctt
3480



 


acactgaaga ttacaataca gtcctctgtc tcctggtatt ccaaagactg ctgctgaatt
3540



 


tctaaagaat agattggtga atgtgactac tcaaagttgt atgtaagact ttcaagggca
3600



 


ttaaataaaa aagaatattg ctg
3623



 









   1
M  D  K  L  R  M  V  L  H  E  N  S  G  S  A  D  F  R  R  C
SEQ ID


   1
atggacaaactcagaatggtgctacatgaaaactctggatctgcagactttagaagatgt
NO: 13


 


  21
S  A  H  L  S  S  F  T  F  A  V  V  A  V  L  S  A  C L   V
and 14


  61
tctgcccatttaagttccttcacttttgctgtagtcgctgttctcagtgcctgcttggtc



 


  41
T  S  S  L  G  G  K  D  K  E  L  R  L  T  G  G  E  N  K  C



 121
actagttctcttggaggaaaagacaaggagctgaggctaacgggtggtgaaaacaagtgc



 


  61
S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W



 181
tctggaagagtggaggtgaaagtgcaggaggagtggggaactgtgtgtaataatggctgg



 


  81
D  M  D  V  V  S  V  V  C  R  Q  L  G  C  P  T  A  I  K  A



 241
gacatggatgtggtctctgttgtttgtaggcagctgggatgtccaactgctatcaaagcc



 


 101
T  G  W  A  N  F  S  A  G  S  G  R  I  W  M  D  H  V  S  C



 301
actggatgggctaattttagtgcaggttctggacgcatttggatggatcatgtttcttgt



 


 121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C



 361
cgagggaatgagtcagctctctgggactgcaaacatgatggatggggaaagcataactgt



 


 141
T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  G  L



 421
actcaccaacaggatgctggagtaacctgctcagatggatctgatttagagatggggctg



 


 161
V  N  G  G  N  R  C  L  G  R  I  E  V  K  F  Q  G  K  W  G



 481
gtgaatggaggaaaccggtgcttaggaagaatagaagtcaaatttcaaggacggtgggga



 


 181
T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L  E



 541
acagtgtgtgatgataacttcaacataaatcatgcttctgtggtttgtaaacaacttgaa



 


 201
C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P  I



 601
tgtggaagtgctgtcagtttctctggttcagctaattttggagaaggttctggaccaatc



 


 221
W  F  D  D  L  V  C  N  G  N  E  S  A  L  W  N  C  K  H  E



 661
tggtttgatgatcttgtatgcaatggaaatgagtcagctctctggaactgcaaacatgaa



 


 241
G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  L  N  G



 721
ggatggggaaagcacaattgcgatcatgctgaggatgctggagtgatttgcttaaatgga



 


 261
A  D  L  K  L  R  V  V  D  G  V  T  E  C  S  G  R  L  E  V



 781
gcagacctgaaactgagagtggtagatggagtcactgaatgttcaggaagattggaagtg



 


 281
K  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  D  D  A  A



 841
aaattccaaggagaatggggaacaatctgtgatgatggctgggatagtgatgatgccgct



 


 301
V  A  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N  A



 901
gtggcatgtaagcaactgggatgtccaactgctgtcactgccattggtcgagttaacgcc



 


 321
S  E  G  T  G  H  I  W  L  D  S  V  S  C  H  G  H  E  S  A



 961
agtgagggaactggacacatttggcttgacagtgtttcttgccatggacacgagtctgct



 


 341
L  W  Q  C  R  H  H  E  W  G  K  H  Y  C  N  H  D  E  D  A



1021
ctctggcagtgtagacaccatgaatggggaaagcattattgcaatcatgatgaagatgct



 


 361
G  V  T  C  S  D  G  S  D  L  E  L  R  L  K  G  G  G  S  H



1081
ggtgtgacatgttctgatggatcagatctggaactgagacttaaaggtggaggcagccac



 


 381
C  A  G  T  V  E  V  E  I  Q  K  L  V  G  K  V  C  D  R  S



1141
tgtgctgggacagtggaggtggaaattcagaaactggtaggaaaagtgtgtgatagaagc



 


 401
W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L  K



1201
tggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatctgcactcaaa



 


 421
T  S  Y  Q  V  Y  S  K  T  K  A  T  N  T  W  L  F  V  S  S



1261
acatcatatcaagtttattccaaaaccaaggcaacaaacacatggctgtttgtaagcagc



 


 441
C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L  S



1321
tgtaatggaaatgaaacttctctttgggactgcaagaattggcagtggggtggacttagt



 


 461
C  D  H  Y  D  E  A  K  I  T  C  S  A  H  R  K  P  R  L  V



1381
tgtgatcactatgacgaagccaaaattacctgctcagcccacaggaaacccaggctggtt



 


 481
G  G  D  I  P  C  S  G  R  V  E  V  Q  H  G  D  T  W  G  T



1441
ggaggggacattccctgctctggtcgtgttgaagtacaacatggagacacgtggggcacc



 


 501
V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q  C



1501
gtctgtgattctgacttctctctggaggcggccagcgtgctgtgcagggaactacagtgc



 


 521
G  T  V  V  S  L  L  G  G  A  H  F  G  E  G  S  G  Q  I  W



1561
ggcactgtggtttccctcctggggggagctcactttggagaaggaagtggacagatctgg



 


 541
A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A  P



1621
gctgaagaattccagtgtgaggggcacgagtcccacctttcactctgcccagtagcaccc



 


 561
R  P  D  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y  T



1681
cgccctgacgggacatgtagccacagcagggacgtcggcgtagtctgctcaagatacaca



 


 581
Q  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  N  I  L



1741
caaatccgcttggtgaatggcaagaccccatgtgaaggaagagtggagctcaacattctt



 


 601
G  S  W  G  S  L  C  N  S  H  W  D  M  E  D  A  H  V  L  C



1801
gggtcctgggggtccctctgcaactctcactgggacatggaagatgcccatgttttatgc



 


 621
Q  Q  L  K  C  G  V  A  L  S  I  P  G  G  A  P  F  G  K  G



1861
cagcagcttaaatgtggagttgccctttctatcccgggaggagcaccttttgggaaagga



 


 641
S  E  Q  V  W  R  H  M  F  H  C  T  G  T  E  K  H  M  G  D



1921
agtgagcaggtctggaggcacatgtttcactgcactgggactgagaagcacatgggagat



 


 661
C  S  V  T  A  L  G  A  S  L  C  S  S  G  Q  V  A  S  V  I



1981
tgttccgtcactgctctgggcgcatcactctgttcttcagggcaagtggcctctgtaatc



 


 681
C  S  G  N  Q  S  Q  T  L  S  P  C  N  S  S  S  S  D  P  S



2041
tgctcagggaaccagagtcagacactatctccgtgcaattcatcatcctcggacccatca



 


 701
S  S  I  I  S  E  E  N  G  V  A  C  I  G  S  G  Q  L  R  L



2101
agctctattatttcagaagaaaatggtgttgcctgcatagggagtggtcaacttcgcctg



 


 721
V  D  G  G  G  R  C  A  G  R  V  E  V  Y  H  E  G  S  W  G



2161
gtcgatggaggtggtcgttgtgctgggagagtagaggtctatcatgagggctcctggggc



 


 741
T  I  C  D  D  S  W  D  L  N  D  A  H  V  V  C  K  Q  L  S



2221
accatctgtgatgacagctgggacctgaatgatgcccatgtggtgtgcaaacagctgagc



 


 761
C  G  W  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P  I



2281
tgtggatgggccattaatgccactggttctgctcattttggggaaggaacagggcccatt



 


 781
W  L  D  E  I  N  C  N  G  K  E  S  H  I  W  Q  C  H  S  H



2341
tggctggatgagataaactgtaatggaaaagaatctcatatttggcaatgccactcacat



 


 801
G  W  G  R  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E  F



2401
ggttgggggcggcacaattgcaggcataaggaggatgcaggagtcatctgctcagagttc



 


 821
M  S  L  R  L  I  S  E  N  S  R  E  T  C  A  G  R  L  E  V



2461
atgtctctgagactgatcagtgaaaacagcagagagacctgtgcagggcgcctggaagtt



 


 841
F  Y  N  G  A  W  G  S  V  G  R  N  S  M  S  P  A  T  V  G



2521
ttttacaacggagcttggggcagcgttggcaggaatagcatgtctccagccacagtgggg



 


 861
V  V  C  R  Q  L  G  C  A  D  R  G  D  I  S  P  A  S  S  D



2581
gtggtatgcaggcagctgggctgtgcagacagaggggacatcagccctgcatcttcagac



 


 881
K  T  V  S  R  H  M  W  V  D  N  V  Q  C  P  K  G  P  D  T



2641
aagacagtgtccaggcacatgtgggtggacaatgttcagtgtcctaaaggacctgacaca



 


 901
L  W  Q  C  P  S  S  P  W  K  K  R  L  A  S  P  S  E  E  T



2701
ctatggcagtgcccatcatctccatggaagaagagactggccagcccctcagaggagaca



 


 921
W  I  T  C  A  N  K  I  R  L  Q  E  G  N  T  N  C  S  G  R



2761
tggatcacatgtgccaacaaaataagacttcaagaaggaaacactaattgttctggacgt



 


 941
V  E  I  W  Y  G  G  S  W  G  T  V  C  D  D  S  W  D  L  E



2821
gtggagatctggtacggaggttcctggggcactgtgtgtgacgactcctgggaccttgaa



 


 961
D  A  Q  V  V  C  R  Q  L  G  C  G  S  A  L  E  A  G  K  E



2881
gatgctcaggtggtgtgccgacagctgggctgtggctcagctttggaggcaggaaaagag



 


 981
A  A  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G  N



2941
gccgcatttggccaggggactgggcccatatggctcaatgaagtgaagtgcaaggggaat



 


1001
E  T  S  L  W  D  C  P  A  R  S  W  G  H  S  D  C  G  H  K



3001
gaaacctccttgtgggattgtcctgccagatcctggggccacagtgactgtggacacaag



 


1021
E  D  A  A  V  T  C  S  E  I  A  K  S  R  E  S  L  H  A  T



3061
gaggatgctgctgtgacgtgctcagaaattgcaaagagccgagaatccctacatgccaca



 


1041
G  R  S  S  F  V  A  L  A  I  F  G  V  I  L  L  A  C  L  I



3121
ggtcgctcatcttttgttgcacttgcaatctttggggtcattctgttggcctgtctcatc



 


1061
A  F  L  I  W  T  Q  K  R  R  Q  R  Q  R  L  S  V  F  S  G



3181
gcattcctcatttggactcagaagcgaagacagaggcagcggctctcagttttctcagga



 


1081
G  E  N  S  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  K  A  D



3241
ggagagaattctgtccatcaaattcaataccgggagatgaattcttgcctgaaagcagat



 


1101
E  T  D  M  L  N  P  S  G  D  H  S  E  V  Q



3301
gaaacggatatgctaaatccctcaggagaccactctgacctacaa



 


   1
MDKLRMVLHE NSGSADFRRC SAHLSSFTFA VVAVLSACLV TSSLGGKDKE
SEQ ID


 


  51
LRLTGGENKC SGRVEVKVQE EWGTVCNNGW DMDVVSVVCR QLGCPTAIKA
NO: 14


 


 101
TGWANFSAGS GRIWMDHVSC RGNESALWDC KHDGWGKHNC THQQDAGVTC



 


 151
SDGSDLEMGL VNGGNRCLGR IEVKFQGRWG TVCDDNFNIN HASVVCKQLE 



 


 201
CGSAVSFSGS ANFGEGSGPI WFDDLVCNGN ESALWNCKHE GWGKHNCDHA



 


 251
EDAGVICLNG ADLKLRVVDG VTECSGRLEV KFQGEWGTIC DDGWDSDDAA



 


 301
VACKQLGCPT AVTAIGRVNA SEGTGHIWLD SVSCHGHESA LWQCRHHEWG



 


 351
KHYCNHDEDA GVTCSDGSDL ELRLKGGGSH CAGTVEVEIQ KLVGKVCDRS



 


 401
WGLKEADVVC RQLGCGSALK TSYQVYSKTK ATNTWLFVSS CNGNETSLWD



 


 451
CKNWQWGGLS CDHYDEAKIT CSAHRKPRLV GGDIPCSGRV EVQHGDTWGT



 


 501
VCDSDFSLEA ASVLCRELQC GTVVSLLGGA HFGEGSGQIW AEEFQCEGHE



 


 551
SHLSLCPVAP RPDGTCSHSR DVGVVCSRYT QIRLVNGKTP CEGRVELNIL



 


 601
GSWGSLCNSH WDMEDAHVLC QQLKCGVALS IPGGAPFGKG SEQVWRHMFH



 


 651
CTGTEKHMGD CSVTALGASL CSSGQVASVI CSGNQSQTLS PCNSSSSDPS



 


 701
SSIISEENGV ACIGSGQLRL VDGGGRCAGR VEVYHEGSWG TICDDSWDLN



 


 751
DAHVVCKQLS CGWAINATGS AHFGEGTGPI WLDEINCNGK ESHIWQCHSH



 


 801
GWGRHNCRHK EDAGVICSEF MSLRLISENS RETCAGRLEV FYNGAWGSVG



 


 851
RNSMSPATVG VVCRQLGCAD RGDISPASSD KTVSRHMWVD NVQCPKGPDT



 


 901
LWQCPSSPWK KRLASPSEET WITCANKIRL QEGNTNCSGR VEIWYGGSWG



 


 951
TVCDDSWDLE DAQVVCRQLG CGSALEAGKE AAFGQGTGPI WLNEVKCKGN



 


1001
ETSLWDCPAR SWGHSDCGHK EDAAVTCSEI AKSRESLHAT GRSSFVALAI



 


1051
FGVILLACLI AFLIWTQKRR QRQRLSVFSG GENSVHQIQY REMNSCLKAD



 


1101
ETDMLNPSGD HSEVQ









Sus CD163 v2 in pCRsusCD163v2 was liberated from pCR2.1 vector after restriction enzymes Kpn I and Not I digestion and gel purification. Recipient vector pCMV-script was also cut with the same restriction enzyme pair and allowed for directional cloning of susCD163v2 into the pCMV-script. After ligation of susCD163 v2 with pCMV-script, the ligated mixture was used to transform STBL 2 E. coli cells (Invitrogen). One transformant was found to contain the CD163 gene by restriction enzyme digestion analysis and was designed pCMV-script susCD163v2 clone#3.
Example 5
Preparation of a RSV Promoter Based Expression System by Direct Ligation and Transfection Method
A non-cloning based procedure to generate microgram quantities of linear DNA suitable for use in generating stable cell lines expressing CD163 from an RSV promoter was developed (FIG. 4). The procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and RSV promoter cassette derived from pRSV-script, and the other containing the susCD163v2 coding sequence from pCMVsusCD163v2. Vector plasmid pRSV-Script was linearized with DraIII upstream of the neomycin gene, and bunted with the Klenow fragment of E. coli DNA polymerase. This plasmid was then digested with NotI immediately downstream of the RSV promoter. The pCMVsusCD163v2 clone was digested in the vector sequence downstream of the CD163 insert with DrdI, and blunted with Klenow fragment of DNA polymerase. The CD163 coding sequence was liberated from the vector with a NotI located immediately upstream of the CD163 coding sequence. For each plasmid digestion the appropriate fragments were purified from agarose gels. A large-scale ligation reaction was performed as follows. Approximately 20 μg of each DNA fragment was incubated in a volume of 600 μl with 15 units of T4 DNA ligase. The reaction was incubated at room temperature for 20 minutes, at which time an aliquot was removed and the reaction frozen on dry ice. Agarose gel analysis of the aliquot revealed that a significant amount of non-ligated DNA remained, so another 15 units of ligase was added and incubated for another 10 minutes at room temperature. Following ligation, a linear piece of DNA containing all of the appropriate elements was purified by agarose gel electrophoresis. Ligation of the two DNA fragments via the cohesive Not I termini resulted in the placement of the 5′ sequences of the CD163 gene downstream of the RSV promoter, allowing for directed expression of CD163 in mammalian cells. Once isolated, the purified DNA was used to transfect various mammalian cell lines.
Example 6
Cloning and Characterization of Human CD163 cDNA
Based on a known human CD163 cDNA sequence (GenBank Accession No. BC051281), a forward primer Hu5′Not (SEQ ID NO: 15) (5′CACCGCGGCCGCGAAGTTATAAATCGCCACCATGAGCAAACTCAGAAT GG-3′) and a reverse primer Hu3′Kpn (SEQ ID NO: 16) (5′-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were designed using the PrimerSelect program. Restriction sites for NotI and KpnI (underlined) were incorporated into the 5′ and 3′ primers, respectively, to facilitate cloning into expression vectors. The sequence CACC was added to the 5′ end of the 5′ primer to allow directional cloning into the pCDNA3.1DN5/V5/His/TOPO vector (Cat. No. K49001, Invitrogen, see FIG. 6). Human CD163 cDNAs were amplified from RNA extracted from the U937 cell line after stimulated with phorbol 12-myristate 13-acetate (100 ng/ml) for 3 days. Total cellular RNA was prepared using the RNeasy kit (Qiagen). RT-PCR reactions and sequencing methods were the same as described in Example 4. PCR products were separated on 0.8% SeaKem agarose gel and extracted from the gel using the GeneClean kit. PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector following the manufacturer's instructions. Two clones with large inserts were sequenced. Sequencing and sequence analysis methods were described in Example 4. A clone with a correct insert was designed “pcDNA3.1D-humCD163v2” and we have designated the sequence of the insert SEQ ID NO: 17
The CD163 open reading frame in pCDNA3.1D-humCD163v2 is 1121 residues in length (designated SEQ ID NO: 18 which encodes SEQ ID NO:19 disclosed below), and is 100% identical to Genbank Z22968 (a human CD163 cDNA of the same length). Our human CD163v2 sequence is also 100% identical to Genbank BC051281 and Z22969 (splice variants of human CD163) except that 42 nonhomologous residues in the two Genbank sequences replace the seven carboxy-terminal residues of our sequence. This difference is due to the presence of an 83-nucleotide exon in BC051281 and Z22969, and the resulting frame shift at the 3′ end of the exon. (Law, S. K., Micklem, K. J., Shaw, J. M., Zhang, X. P., Dong, Y., Willis, A. C. and Mason, D. Y. (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. European Journal of Immunology 23 (9), 2320-2325).












SEQUENCE

ID NO







atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgactt cagaagacat
  60
SEQ ID


 


tttgtcaacc tgagtccctt caccattact gtggtcttac ttctcagtgc ctgttttgtc
 120
ID: 17


 


accagttctc ttggaggaac agacaaggag ctgaggctag tggatggtga aaacaagtgt
 180



 


agcgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg
 240



 


agcatggaag cggtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc
 300



 


cctggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt
 360



 


cgtgggaatg agtcagctct ttgggattgc aaacatgatg gatggggaaa gcatagtaac
 420



 


tgtactcacc aacaagatgc tggagtgacc tgctcagatg gatccaattt ggaaatgagg
 480



 


ctgacgcgtg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacggtgg
 540



 


ggaacagtgt gtgatgataa cttcaacata gatcatgcat ctgtcatttg tagacaactt
 600



 


gaatgtggaa gtgctgtcag tttctctggt tcatctaatt ttggagaagg ctctggacca
 660



 


atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat
 720



 


caaggatggg gaaagcataa ctgtgaccat gctgaggatg ctggagtgat ttgctcaaag
 780



 


ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa
 840



 


gtgagattcc aaggggaatg ggggacaata tgtgatgacg gctgggacag ttacgatgct
 900



 


gctgtggcat gcaagcaact gggatgtcca actgccgtca cagccattgg tcgagttaac
 960



 


gccagtaagg gatttggaca catctggctt gacagcgttt cttgccaggg acatgaacct
1020



 


gctgtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat
1080



 


gctggcgtga catgttctga tggatcagat ciggagctaa gacttagagg tggaggcagc
1140



 


cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga
1200



 


ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc
1260



 


aaaacatctt atcaagtgta ctccaaaatc caggcaacaa acacatggct gtttctaagt
1320



 


agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt
1380



 


acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg
1440



 


gttggagggg acattccctg ttctggacgt gttgaagtga agcatggtga cacgtggggc
1500



 


tccatctgtg attcggactt ctctctggaa gctgccagcg ttctatgcag ggaattacag
1560



 


tgtggcacag ttgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc
1620



 


tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca
1680



 


ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac
1740



 


acagaaattc gcttggtgaa tggcaagacc ccgtgtgagg gcagagtgga gctcaaaacg
1800



 


cttggtgcct ggggatccct ctgtaactct cactgggaca tagaagatgc ccatgttctt
1860



 


tgccagcagc ttaaatgtgg agttgccctt tctaccccag gaggagcacg ttttggaaaa
1920



 


ggaaatggtc agatctggag gcatatgttt cactgcactg ggactgagca gcacatggga
1980



 


gattgtcctg taactgctct aggtgcttca ttatgtcctt cagagcaagt ggcctctgta
2040



 


atctgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcgtc tttgggccca
2100



 


acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc
2160



 


ctggtaaatg gaggaggtcg ctgtgctggg agagtagaga tctatcatga gggctcctgg
2220



 


ggcaccatct gtgatgacag ctgggacctg agtgatgccc acgtggtttg cagacagctg
2280



 


ggctgtggag aggccattaa tgccactggt tctgctcatt ttggggaagg aacagggccc
2340



 


atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca
2400



 


cacggctggg ggcagcaaaa ttgcaggcac aaggaggatg cgggagttat ctgctcagaa
2460



 


ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctggaa
2520



 


gttttttaca atggagcttg gggcactgtt ggcaagagta gcatgtctga aaccactgtg
2580



 


ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta
2640



 


gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac
2700



 


acgctgtggc agtgcccatc atctccatgg gagaagagac tggccagccc ctcggaggag
2760



 


acctggatca catgtgacaa caagataaga cttcaggaag gacccacttc ctgttctgga
2820



 


cgtgtggaga tctggcatgg aggttcctgg gggacagtgt gtgatgactc ttgggacttg
2880



 


gacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa
2940



 


gaagcagagt ttggtcaggg gactggaccg atatggctca atgaagtgaa gtgcaaaggg
3000



 


aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccatagtga gtgtgggcac
3060



 


aaggaagacg ctgcagtgaa ttgcacagat atttcagtgc agaaaacccc acaaaaagcc
3120



 


acaacaggtc gctcatcccg tcagtcatcc tttattgcag tcgggatcct tggggttgtt
3180



 


ctgttggcca ttttcgtcgc attattcttc ttgactaaaa agcgaagaca gagacagcgg
3240



 


cttgcagttt cctcaagagg agagaactta gtccaccaaa ttcaataccg ggagatgaat
3300



 


tcttgcctga atgcagatga tctggaccta atgaattcct caggaggcca ttctgagcca
3360



 


cactgaaaag gaaaatggga atttataacc cagtgagttc agcctttaag ataccttgat
3420



 


gaagacctgg acta
3434



 









   1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  F  R  R  H
SEQ ID


   1
atgagcaaactcagaatggtgctacttgaagactctggatctgctgacttcagaagacat
NO: 18


 


  21
F  V  N  L  S  P  F  T  I  T  V  V  L  L  L  S  A  C  F  V
and 19


  61
tttgtcaacctgagtcccttcaccattactgtggtcttacttctcagtgcctgttttgtc



 


  41
T  S  S  L  G  G  T  D  K  E  L  R  L  V  D  G  E  N  K  C



 121
accagttctcttggaggaacagacaaggagctgaggctagtggatggtgaaaacaagtgt



 


  61
S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N G   W



 181
agcgggagagtggaagtgaaagtccaggaggagtggggaacggtgtgtaataatggctgg



 


  81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



 241
agcatggaagcggtctctgtgatttgtaaccagctgggatgtccaactgctatcaaagcc



 


 101
P  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



 301
cctggatgggctaattccagtgcaggttctggacgcatttggatggatcatgtttcttgt



 


 121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



 361
cgtgggaatgagtcagctctttgggattgcaaacatgatggatggggaaagcatagtaac



 


 141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  N  L  E  M  R



 421
tgtactcaccaacaagatgctggagtgacctgctcagatggatccaatttggaaatgagg



 


 161
L  T  R  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  R  W



 481
ctgacgcgtggagggaatatgtgttctggaagaatagagatcaaattccaaggacggtgg



 


 181
G  T  V  C  D  D  N  F  N  I  D  H  A  S  V  I  C  R  Q  L



 541
ggaacagtgtgtgatgataacttcaacatagatcatgcatctgtcatttgtagacaactt



 


 201
E  C  G  S  A  V  S  F  S  G  S  S  N  F  G  E  G  S  G  P



 601
gaatgtggaagtgctgtcagtttctctggttcatctaattttggagaaggctctggacca



 


 221
I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



 661
atctggtttgatgatcttatatgcaacggaaatgagtcagctctctggaactgcaaacat



 


 241
Q  G  W  K  H  N  C  H  D  H  A  E  D  A  G  V  I  C  S  K



 721
caaggatggggaaagcataactgtgaccatgctgaggatgctggagtgatttgctcaaag



 


 261
G  A  D  L  S  L  K  L  V  D  G  V  T  E  C  S  G  R  L  E



 781
ggagcagatctgagcctgagactggtagatggagtcactgaatgttcaggaagattagaa



 


 281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  Y  D  A



 841
gtgagattccaaggggaatgggggacaatatgtgatgacggctgggacagttacgatgct



 


 301
A  V  A  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N



 901
gctgtggcatgcaagcaactgggatgtccaactgccgtcacagccattggtcgagttaac



 


 321
A  S  K  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



 961
gccagtaagggatttggacacatctggcttgacagcgtttcttgccagggacatgaacct



 


 341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
gctgtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat



 


 361
A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S



1081
gctggcgtgacatgttctgatggatcagatctggagctaagacttagaggtggaggcagc



 


 381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
cgctgtgctgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga



 


 401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
ggctggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatctgcactc



 


 421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  T  W  L  F  L  S



1261
aaaacatcttatcaagtgtactccaaaatccaggcaacaaacacatggctgtttctaagt



 


 441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
agctgtaacggaaatgaaacttctctttgggactgcaagaactggcaatggggtggactt



 


 461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  A  L



1381
acctgtgatcactatgaagaagccaaaattacctgctcagcccacagggaacccagactg



 


 481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
gttggaggggacattccctgttctggacgtgttgaagtgaagcatggtgacacgtggggc



 


 501
S  I  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
tccatctgtgattcggacttctctctggaagctgccagcgttctatgcagggaattacag



 


 521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
tgtggcacagttgtctctatcctggggggagctcactttggagagggaaatggacagatc



 


 541
W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
tgggctgaagaattccagtgtgagggacatgagtcccatctttcactctgcccagtagca



 


 561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
ccccgcccagaaggaacttgtagccacagcagggatgttggagtagtctgctcaagatac



 


 581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
acagaaattcgcttggtgaatggcaagaccccgtgtgagggcagagtggagctcaaaacg



 


 601
L  G  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
cttggtgcctggggatccctctgtaactctcactgggacatagaagatgcccatgttctt



 


 621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  R  F  G  K



1861
tgccagcagcttaaatgtggagttgccctttctaccccaggaggagcacgttttggaaaa



 


 641
G  N  G  Q  I  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
ggaaatggtcagatctggaggcatatgtttcactgcactgggactgagcagcacatggga



 


 661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  E  Q  V  A  S  V



1981
gattgtcctgtaactgctctaggtgcttcattatgtccttcagagcaagtggcctctgta



 


 681
I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041
atctgctcaggaaaccagtcccaaacactgtcctcgtgcaattcatcgtctttgggccca



 


 701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
acaaggcctaccattccagaagaaagtgctgtggcctgcatagagagtggtcaacttcgc



 


 721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161
ctggtaaatggaggaggtcgctgtgctgggagagtagagatctatcatgagggctcctgg



 


 741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
ggcaccatctgtgatgacagctgggacctgagtgatgcccacgtggtttgcagacagctg



 


 761
G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
ggctgtggagaggccattaatgccactggttctgctcattttggggaaggaacagggccc



 


 781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
atctggctggatgagatgaaatgcaatggaaaagaatcccgcatttggcagtgccattca



 


 801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
cacggctgggggcagcaaaattgcaggcacaaggaggatgcgggagttatctgctcagaa



 


 821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
ttcatgtctctgagactgaccagtgaagccagcagagaggcctgtgcagggcgtctggaa



 


 841
V  F  Y  N  G  A  W  G  T  V  G  K  S  S  M  S  E  T  T  V



2521
gttttttacaatggagcttggggcactgttggcaagagtagcatgtctgaaaccactgtg



 


 861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  P  A  S  L



2581
ggtgtggtgtgcaggcagctgggctgtgcagacaaagggaaaatcaaccctgcatcttta



 


 881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
gacaaggccatgtccattcccatgtgggtggacaatgttcagtgtccaaaaggacctgac



 


 901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  S  P  S  E  E



2701
acgctgtggcagtgcccatcatctccatgggagaagagactggccagcccctcggaggag



 


 921
T  W  I  T  C  D  N  K  I  R  L  Q  E  G  P  T  S  C  S  G



2761
acctggatcacatgtgacaacaagataagacttcaggaaggacccacttcctgttctgga



 


 941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
cgtgtggagatctggcatggaggttcctgggggacagtgtgtgatgactcttgggacttg



 


 961
D  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
gacgatgctcaggtggtgtgtcaacaacttggctgtggtccagctttgaaagcattcaaa



 


 981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941
gaagcagagtttggtcaggggactggaccgatatggctcaatgaagtgaagtgcaaaggg



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
aatgagtcttccttgtgggattgtcctgccagacgctggggccatagtgagtgtgggcac



 


1021
K  E  D  A  A  V  N  C  T  D  I  S  V  Q  K  T  P  Q  K  A



3061
aaggaagacgctgcagtgaattgcacagatatttcagtgcagaaaaccccacaaaaagcc



 


1041
T  T  G  R  S  S  R  Q  S  S  F  I  A  V  G  I  L  G  V  V



3121
acaacaggtcgctcatcccgtcagtcatcctttattgcagtcgggatccttggggttgtt



 


1061
L  L  A  I  F  V  A  L  F  F  L  T  K  K  A  R  Q  R  Q  R



3181
ctgttggccattttcgtcgcattattcttcttgactaaaaagcgaagacagagacagcgg



 


1081
L  A  V  S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N



3241
cttgcagtttcctcaagaggagagaacttagtccaccaaattcaataccgggagatgaat



 


1101
S  C  L  N  A  D  D  L  D  L  M  N  S  S  G  G  H  S  E  P



3301
tcttgcctgaatgcagatgatctggacctaatgaattcctcaggaggccattctgagcca



 


1121
H



3361 
cac



 


   1
MSKLRMVLLE DSGSADFRRH FVNLSPFTIT VVLLLSACFV TSSLGGTDKE
SEE ID


 


  51
LRLVDGENKC SGRVEVKVQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA
NO: 19


 


 101
PGWANSSAGS GRIWMDHVSC RGNESALWDC KHDGWGKHSN CTHQQDAGVT



 


 151
CSDGSNLEMR LTRGGNMCSG RIEIKFQGRW GTVCDDNFNI DHASVICRQL



 


 201
ECGSAVSFSG SSNFGEGSGP IWFDDLICNG NESALWNCKH QGWGKHNCDH



 


 251
AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSYDA



 


 301
AVACKQLGCP TAVTAIGRVN ASKGFGHIWL DSVSCQGHEP AVWQCKHHEW



 


 351
GKHYCNHNED AGVICSOGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR



 


 401
GWGLKEADVV CRQLGCGSAL KTSYQVYSKI QATNTWLFLS SCNGNETSLW



 


 451
DCKNWQWGGL TCDRYEEAKI TCSAHREPRL VGGDIPCSGR VEVKHGDTWG



 


 501
SICDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI WAEEFQCEGH



 


 551
ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



 


 601
LGAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGARFGK GNGQIWRHMF



 


 651
HCTGTEQHMG DCPVTALGAS LCPSEQVASV ICSGNQSQTL SSCNSSSLGP



 


 701
TRPTIPEESA VACIESGQLR LVNGGGRCAG RVEIYHEGSW GTICDDSWDL



 


 751
SDAHVVCRQL GCGEAINATG SAHFGEGTGP IWLDEMKCNG KESRIWQCHS



 


 801
HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE VFYNGAWGTV



 


 851
GKSSMSETTV GVVCRQLGCA DKGKINPASL DKAMSIPMWV DNVQCPKGPD



 


 901
TLWQCPSSPW EKRLASPSEE TWITCDNKIR LQEGPTSCSG RVEIWHGGSW



 


 951
GTVCDDSWDL DDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG



 


1001
NESSLWDCPA RRWGHSECGH KIIOAVNCTD ISVQKTPQKA TTGRSSRQSS



 


1051
FIAVGILGVV LLAIFVALFF LTKKRRQRQR LAVSSRGENL VHQIQYREMN



 


1101
SCLNADDLDL MNSSGGHSEP H









Example 7
Cloning and Characterization of Murine CD163
Based on the murine CD163 sequence in GenBank (AF274883), a forward primer Mus-new5′ (SEQ ID NO: 20) (5′-CACCGCGGCCGCCACACGGAGCCATCAAAATCATCAA-3′) and a reverse primer Mus-new3′ (SEQ ID NO:21) (5′-GGTACCGCGAACAAGCAAACCAATAGCAATATTGTTTAATTCCCTC-3′) were designed using the PrimerSelect program. Restriction endonucleases sites for NotI and KpnI were included in 5′ and 3′ primers, respectively, to allow future cloning into other expression vectors. Mouse peritoneal macrophages were harvested from mice 2 days after injecting thioglycollate medium into the peritoneal cavity. Total cellular RNA was prepared from peritoneal macrophages using the RNeasy kit. RT-PCR reactions and RT-PCR parameters were the same as described in Example 4, except the annealing temperature was increased to 60° C. and extension temperature increased to 72° C. The PCR product was purified on a 0.8% SeaKem agarose gel and directionally cloned into pCDNA3.1D/V5/His/TOPO according to the manufacturer's instructions. Several clones with large inserts were identified for further analysis. A plasmid containing an insert (SEQ ID NO: 22) with a murine CD163 that encodes a protein of the same length as (1121 amino acids SEQ ID NO:24) and differs from Genbank AF274883 by only two amino acids (99.8% identity) was designated “pCDNA3.1D-murCD163v2”.
Another plasmid, “pCDNA3.1D-murCD163v3 was generated which contained an insert (SEQ ID NO: 25) containing a murine CD163 coding sequence (SEQ ID NO: 26) which encodes a protein of 1159 amino acids in length (SEQ ID NO: 27). It differs from AF274883 by only 3 amino acids within the first 1107 residues (99.7% identity), but the sequences diverge completely beginning at residue 1108. This is due to an insertion of 82 nucleotides in the cDNA, and a concomitant shift in reading frame downstream of the insertion. As a result, murine CD163v3 contains 52 amino acids at its carboxy-terminus that are not homologous to the 14 carboxy-terminal residues of murine CD163v2. These two alternative versions of “full length” murine CD163 are most likely splice variant of the same gene, as has been described for human CD163 (Law, S. K., Micklem, K. J., Shaw, J. M., Zhang, X. P., Dong, Y., Willis, A. C. and Mason, D. Y. (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. European Journal of Immunology 23 (9), 2320-2325).












SEQUENCE

ID NO







gctttggaat gggtggacac agaatggttc ttcttggagg tgctggatct cctggttgta
  60
SEQ ID


 


aaaggtttgt ccatctaggt ttctttgttg tggctgtgag ctcacttctc agtgcctctg
 120
NO: 22


 


ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggcgggt ggtgaaaaca
 180



 


actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg
 240



 


gctggagcat gaatgaagtg tccgtggttt gccagcagct gggatgccca acttctatta
 300



 


aagcccttgg atgggctaac tccagcgccg gctctggata tatctggatg gacaaagttt
 360



 


cttgtacagg gaatgagtca gctctttggg actgcaaaca tgatgggtgg ggaaagcata
 420



 


actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga
 480



 


gactggtgaa cagtgcgggc caccgatgct taggaagagt agaaataaag ttccagggaa
 540



 


agtgggggac ggtgtgtgac gacaacttca gcaaagatca cgcttctgtg atttgtaaac
 600



 


agcttggatg tggaagtgcc attagtttct ctggctcagc taaattggga gctggttctg
 660



 


gaccaatctg gctcgatgac ctggcatgca atggaaatga gtcagctctc tgggactgca
 720



 


aacaccgggg atggggcaag cataactgtg accatgctga ggatgtcggt gtgatttgct
 780



 


tagagggagc agatctgagc ctgagactag tggatggagt gtccagatgt tcaggaagat
 840



 


tggaagtgag attccaagga gaatggggga ccgtgtgtga tgataactgg gatctccggg
 900



 


atgcttctgt ggtgtgcaag caactgggat gtccaactgc catcagtgcc attggtcgag
 960



 


ttaatgccag tgagggatct ggacagattt ggcttgacaa catttcatgc gaaggacatg
1020



 


aggcaactct ttgggagtgt aaacaccaag agtggggaaa gcattactgt catcatagag
1080



 


aagacgctgg cgtgacatgt tctgatggag cagatctgga acttagactt gtaggtggag
1140



 


gcagtcgctg tgctggcatt gtggaggtgg agattcagaa gctgactggg aagatgtgta
1200



 


gccgaggctg gacactggca gatgcggatg tggtttgcag acagcttgga tgtggatctg
1260



 


cgcttcaaac ccaggctaag atctactcta aaactggggc aacaaatacg tggctctttc
1320



 


ctggatcttg taatggaaat gaaactactt tttggcaatg caaaaactgg cagtggggcg
1380



 


gcctttcctg tgataatttc gaagaagcca aagttacctg ctcaggccac agggaaccca
1440



 


gactggttgg aggagaaatc ccatgctctg gtcgtgtgga agtgaaacac ggagacgtgt
1500



 


ggggctccgt ctgtgatttt gacttgtctc tggaagctgc cagtgtggtg tgcagggaat
1560



 


tacaatgtgg aacagtcgtc tctatcctag ggggagcaca ttttggagaa ggaagtggac
1620



 


agatctgggg tgaagaattc cagtgtagtg gggatgagtc ccatctttca ctatgctcag
1680



 


tggcgccccc gctagacaga acttgtaccc acagcaggga tgtcagcgta gtctgctcac
1740



 


gatacataga tattcgtctg gcaggcggcg agtcctcctg tgagggaaga gtggagctca
1800



 


agacactcgg agcctggggt cccctctgca gttctcattg ggacatggaa gatgctcatg
1860



 


tcttatgtca gcagctgaag tgtggggttg cccaatctat tccagaagga gcacattttg
1920



 


ggaaaggagc tggtcaggtc tggagtcaca tgttccactg cactggaact gaggaacata
1980



 


taggagattg cctcatgact gctctgggtg cgccgacgtg ttccgaagga caggtggcct
2040



 


ctgtcatctg ctcaggaaac caatcccaga cactattgcc atgtagttca ttgtctccag
2100



 


tccaaacaac aagctctaca attccaaagg agagtgaagt tccctgcata gcaagtggcc
2160



 


agcttcgctt ggtaggtgga ggtggtcgct gcgctggaag agtggaggtc taccacgagg
2220



 


gctcttgggg caccgtctgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca
2280



 


agcagctgga ctgtggcgtg gcaattaacg ccactggctc tgcttacttc ggggaaggag
2340



 


caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggcagt
2400



 


gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct
2460



 


gctccgagtt catgtctctg aggctgacca acgaagccca caaagaaaac tgcacaggtc
2520



 


gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtctccaa
2580



 


ccactgtggg ggtggtgtgc cgtcagctgg gctgtgcaga caacgggact gtgaaaccca
2640



 


taccttcaga caagacacca tccaggccca tgtgggtaga tcgtgtgcag tgtccaaaag
2700



 


gagttgacac tttgtggcag tgcccctcgt caccttggaa acagagacag gccagcccct
2760



 


cctcccagga gtcctggatc atctgtgaca acaaaataag actccaggaa gggcatacag
2820



 


actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact
2880



 


cctgggatct taatgatgct aaggttgtat gtaagcagtt gggctgtggc caagctgtga
2940



 


aggcactaaa agaagcagca tttggtccag gaactgggcc catatggctc aatgaaatta
3000



 


agtgtagagg gaatgagtct tccctgtggg attgtcctgc caaaccgtgg agtcacagcg
3060



 


actgtgggca caaagaagat gcttccatcc agtgcctccc aaaaatgact tcagaatcac
3120



 


atcatggcac aggtcacccc accctcacgg cactcttggt ttgtggagcc attctattgg
3180



 


toctcctcat tgtcttcctc ctgtggactc tgaagcgacg acagattcag cgacttacag
3240



 


tttcctcaag aggagaggtc ttgatacatc aagttcagta ccaagagatg gattcaaagg
3300



 


cggatgatct ggacttgctg aaatcctcgg gggtcattca gaggcacact gagaaggaaa
3360



 


atgataattt ataatccact gaggttggag tttaagaagc cttgacagga cagccagcta
3420



 


aatggaacaa gagcccaggc aacgcacgga tgaccacagc tgcatcttca tgcagtcctt
3480



 


tgtttcctgg aactctgctg aacctgcaaa aaccatattt gtgaatgtga ccacttaata
3540



 


gagatgggag actttt
3556



 









   1
M  G  G  H  R  M  V  L  L  G  G  A  G  S  P  G  C  K  R  F
SEQ ID


   1
atgggtggacacagaatggttcttcttggaggtgctggatctcctggttgtaaaaggttt
NO: 23


 


  21
V  H  L  G  F  F  V  V  A  V  S  S  L  L  S  A  S  A  V  T
and 24


  61
gtccatctaggtttctttgttgtggctgtgagctcacttctcagtgcctctgctgtcact



 


  41
N  A  P  G  E  M  K  K  E  L  R  L  A  G  G  E  N  N  C  S



 121
aacgctcctggagaaatgaagaaggaactgagactggcgggtggtgaaaacaactgtagt



 


  61
C  R  V  E  L  K  I  H  D  K  W  G  T  V  C  S  N  G  W  S



 181
gggagagtggaacttaagatccatgacaagtggggcacagtgtgcagtaacggctggagc



 


  81
M  N  E  V  S  V  V  C  Q  Q  L  G  C  P  T  S  I K  A   L



 241
atgaatgaagtgtccgtggtttgccagcagctgggatgcccaacttctattaaagccctt



 


 101
G  W  A  N  S  S  A  G  S  G  Y  I  W  M  D  K  V  S  C  T



 301
ggatgggctaactccagcgccggctctggatatatctggatggacaaagtttcttgtaca



 


 121
G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N C   T



 361
gggaatgagtcagctctttgggactgcaaacatgatgggtggggaaagcataactgtacc



 


 141
H  E  K  D  A  G  V  T  C  S  D  G  S  N  L  E  M  R  L  V



 421
catgaaaaagatgctggagtgacctgctcagatggatctaatttggagatgagactggtg



 


 161
N  S  A  G  H  R  C  L  G  R  V  E  I  K  F  Q  G  K  W  G



 481
aacagtgcgggccaccgatgcttaggaagagtagaaataaagttccagggaaagtggggg



 


 181
T  V  C  D  D  N  F  S  K  D  H  A  S  V  I  C  K  Q  L  G



 541
acggtgtgtgacgacaacttcagcaaagatcacgcttctgtgatttgtaaacagcttgga



 


 201
C G   S  A  I  S  F  S  G  S  A  K  L  G  A  G  S  G  P  I



 601
tgtggaagtgccattagtttctctggctcagctaaattgggagctggttctggaccaatc



 


 221
W  L  D  D  L  A  C  N  G  N  E  S  A  L  W  D  C  K  H  R



 661
tggctcgatgacctggcatgcaatggaaatgagtcagctctctgggactgcaaacaccgg



 


 241
G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  E  G



 721
ggatggggcaagcataactgtgaccatgctgaggatgtcggtgtgatttgcttagaggga



 


 261
A  D  L  S  L  R  L  V  D  G  V  S  R  C  S  G  R  L  E  V



 781
gcagatctgagcctgagactagtggatggagtgtccagatgttcaggaagattggaagtg



 


 281
R  F  Q  G  E  W  G  T  V  C  D  D  N  W  D  L  R  D  A  S



 841
agattccaaggagaatgggggaccgtgtgtgatgataactgggatctccgggatgcttct



 


 301
V  V  C  K  Q  L  G  C  P  T  A  I  S  A  I  G  R  V  N  A



 901
gtggtgtgcaagcaactgggatgtccaactgccatcagtgccattggtcgagttaatgcc



 


 321
S  E  G  S  G  Q  I  W  L  D  N  I  S  C  E  G  H  E  A  T



 961
agtgagggatctggacagatttggcttgacaacatttcatgcgaaggacatgaggcaact



 


 341
L  W  E  C  K  H  Q  E  W  G  K  H  Y  C  H  H  R  E  D  A



1021
ctttgggagtgtaaacaccaagagtggggaaagcattactgtcatcatagagaagacgct



 


 361
G  V  T  C  S  D  G  A  D  L  E  L  R  L  V  G  G  G  S  R



1081
ggcgtgacatgttctgatggagcagatctggaacttagacttgtaggtggaggcagtcgc



 


 381
C  A  G  I  V  E  V  E  I  Q  K  L  T  G  K  M  C  S  R  G



1141
tgtgctggcattgtggaggtggagattcagaagctgactgggaagatgtgtagccgaggc



 


 401
W  T  L  A  D  A  D  V  V  C  R  Q  L  G  C  G  S  A  L  Q



1201
tggacactggcagatgcggatgtggtttgcagacagcttggatgtggatctgcgcttcaa



 


 421
T  Q  A  K  I  Y  S  K  T  G  A  T  N  T  W  L  F  P  G  S



1261
acccaggctaagatctactctaaaactggggcaacaaatacgtggctctttcctggatct



 


 441
C  N  G  N  E  T  T  F  W  Q  C  K  N  W  Q  W  G  G  L  S



1321
tgtaatggaaatgaaactactttttggcaatgcaaaaactggcagtggggcggcctttcc



 


 461
C  D  N  F  E  E  A  K  V  T  C  S  G  H  R  E  P  R  L  V



1381
tgtgataatttcgaagaagccaaagttacctgctcaggccacagggaacccagactggtt



 


 481
G  G  E  I  P  C  S  G  R  V  E  V  K  H  G  D  V  W  G  S



1441
ggaggagaaatcccatgctctggtcgtgtggaagtgaaacacggagacgtgtggggctcc



 


 501
V  C  D  F  D  L  S  L  E  A  A  S  V  V  C  R  E  L  Q  C



1501
gtctgtgattttgacttgtctctggaagctgccagtgtggtgtgcagggaattacaatgt



 


 521
G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  S  G  Q  I  W



1561
ggaacagtcgtctctatcctagggggagcacattttggagaaggaagtggacagatctgg



 


 541
G  E  E  F  Q  C  S  G  D  E  S  H  L  S  L  C  S  V  A  P



1621
ggtgaagaattccagtgtagtggggatgagtcccatctttcactatgctcagtggcgccc



 


 561
P  L  D  R  T  C  T  H  S  R  D  V  S  V  V  C  S  R  Y  I



1681
ccgctagacagaacttgtacccacagcagggatgtcagcgtagtctgctcacgatacata



 


 581
D  I  R  L  A  G  G  E  S  S  C  E  G  R  V  E  L  K  T  L



1741
gatattcgtctggcaggcggcgagtcctcctgtgagggaagagtggagctcaagacactc



 


 601
G  A  W  G  P  L  C  S  S  H  W  D  M  E  D  A  H  V  L  C



1801
ggagcctggggtcccctctgcagttctcattgggacatggaagatgctcatgtcttatgt



 


 621
Q  Q  L  K  C  G  V  A  Q  S  I  P  E  G  A  H  F  G  K  G



1861
cagcagctgaagtgtggggttgcccaatctattccagaaggagcacattttgggaaagga



 


 641
A  G  Q  V  W  S  H  M  F  H  C  T  G  T  E  E  H  I  G  D



1921
gctggtcaggtctggagtcacatgttccactgcactggaactgaggaacatataggagat



 


 661
C  L  M  T  A  L  G  A  P  T  C  S  E  G  Q  V  A  S  V  I  



1981
tgcctcatgactgctctgggtgcgccgacgtgttccgaaggacaggtggcctctgtcatc



 


 681
C  S  G  N  Q  S  Q  T  L  L  P  C  S  S  L  S  P  V  Q  T



2041
tgctcaggaaaccaatcccagacactattgccatgtagttcattgtctccagtccaaaca



 


 701
T  S  S  T  I  P  K  E  S  E  V  P  C  I  A  S  G  Q  L  R



2101
acaagctctacaattccaaaggagagtgaagttccctgcatagcaagtggccagcttcgc



 


 721
L  V  G  G  G  G  R  C  A  G  R  V  E  V  Y  H  E  G  S  W



2161
ttggtaggtggaggtggtcgctgcgctggaagagtggaggtctaccacgagggctcttgg



 


 741
G  T  V  C  D  D  N  W  D  M  T  D  A  N  V  V  C  K  Q  L



2221
ggcaccgtctgtgatgacaattgggatatgactgatgccaatgtggtgtgcaagcagctg



 


 761
D  C  G  V  A  I  N  A  T  G  S  A  Y  F  G  E  G  A  G  A



2281
gactgtggcgtggcaattaacgccactggctctgcttacttcggggaaggagcaggagct



 


 781
I  W  L  D  E  V  I  C  T  G  K  E  S  H  I  W  Q  C  H  S



2341
atctggctagacgaagtcatctgcactgggaaagagtctcatatttggcagtgccattca



 


 801
H  G  W  G  R  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
catggctggggacgccataactgcaggcacaaagaagatgcaggtgttatctgctccgag



 


 821
F  M  S  L  R  L  T  N  E  A  H  K  E  N  C  T  G  R  L  E



2461
ttcatgtctctgaggctgaccaacgaagcccacaaagaaaactgcacaggtcgccttgaa



 


 841
V  F  Y  N  G  T  W  G  S  I  G  S  S  N  M  S  P  T  T  V



2521
gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctccaaccactgtg



 


 861
G  V  V  C  R  Q  L  G  C  A  D  N  G  T  V  K  P  I  P  S



2581
ggggtggtgtgccgtcagctgggctgtgcagacaacgggactgtgaaacccataccttca



 


 881
D  K  T  P  S  R  P  M  W  V  D  R  V  Q  C  P  K  G  V  D



2641
gacaagacaccatccaggcccatgtgggtagatcgtgtgcagtgtccaaaaggagttgac



 


 901
T  L  W  Q  C  P  S  S  P  W  K  Q  R  Q  A  S  P  S  S  Q



2701
actttgtggcagtgcccctcgtcaccttggaaacagagacaggccagcccctcctcccag



 


 921
E  S  W  I  I  C  D  N  K  I  R  L  Q  E  G  H  T  D  C  S



2761
gagtcctggatcatctgtgacaacaaaataagactccaggaagggcatacagactgttct



 


 941
G  R  V  E  I  W  H  K  G  S  W  G  T  V  C  D  D  S  W  D



2821
ggacgtgtggagatctggcacaaaggttcctggggaacagtgtgtgatgactcctgggat



 


 961
L  N  D  A  K  V  V  C  K  Q  L  G  C  G  Q  A  V  K  A  L



2881
cttaatgatgctaaggttgtatgtaagcagttgggctgtggccaagctgtgaaggcacta



 


 981
K  E  A  A  F  G  P  G  T  G  P  I  W  L  N  E  I  K  C  R



2941
aaagaagcagcatttggtccaggaactgggcccatatggctcaatgaaattaagtgtaga



 


1001
G  N  E  S  S  L  W  D  C  P  A  K  P  W  S  H  S  D  C  G



3001
gggaatgagtcttccctgtgggattgtcctgccaaaccgtggagtcacagcgactgtggg



 


1021
H  K  E  D  A  S  I  Q  C  L  P  K  M  T  S  E  S  H  H  G



3061
cacaaagaagatgcttccatccagtgcctcccaaaaatgacttcagaatcacatcatggc



 


1041
T  G  H  P  T  L  T  A  L  L  V  C  G  A  I  L  L  V  L  L



3121
acaggtcaccccaccctcacggcactcttggtttgtggagccattctattggtcctcctc



 


1061
I  V  F  L  L  W  T  L  K  R  R  Q  I  Q  R  L  T  V  S  S



3181
attgtcttcctcctgtggactctgaagcgacgacagattcagcgacttacagtttcctca



 


1081
R  G  E  V  L  I  H  Q  V  Q  Y  Q  E  M  D  S  K  A  D  D



3241
agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggcggatgat



 


1101
L  D  L  L  K  S  S  G  V  I  Q  R  H  T  E  K  E  N  D  N



3301
ctggacttgctgaaatcctcgggggtcattcagaggcacactgagaaggaaaatgataat



 


1121
L



3361
tta



 


   1
MGGHRMVLLG GAGSPGCKRF VHLGFFVVAV SSLLSASAVT NAPGEMKKEL
SEQ ID


 


  51
RLAGGENNCS GRVELKIHDK WGTVCSNGWS MNEVSVVCQQ LGCPTSIKAL
NO: 24


 


 101
GWANSSAGSG YIWMDKVSCT GNESALWDCK HDGWGKHNCT HEKDAGVTCS



 


 151
DGSNLEMRLV NSAGHRCLGR VEIKFQGKWG TVCDDNFSKD HASVICKQLG



 


 201
CGSAISFSGS AKLGAGSGPI WLDDLACNGN ESALWDCKHR GWGKHNCDHA



 


 251
EDVGVICLEG ADLSLRLVDG VSRCSGRLEV RFQGEWGTVC DDNWDLRDAS



 


 301
VVCKQLGCPT AISAIGRVNA SEGSGQIWLD NISCEGHEAT LWECKHQEWG



 


 351
KHYCHEREDA GVTCSDGADL ELRLVGGGSR CAGIVEVEIQ KLTGKMCSRG



 


 401
WTLADADVVC RQLGCGSALQ TQAKIYSKTG ATNTWLFPGS CNGNETTFWQ



 


 451
CKNWQWGGLS CDNEFEAKVT CSGHREPRLV GGEIPCSGRV EVKHGDVWGS



 


 501
VCDFDLSLEA ASVVCRELQC GTVVSILGGA HFGEGSGQIW GEEFQCSGDE



 


 551
SHLSLCSVAP PLDRTCTHSR DVSVVCSRYI DIRLAGGESS CEGRVELKTL



 


 601
GAWGPLCSSH WDMEDAHVLC QQLKCGVAQS IPEGAHFGKG AGQVWSHMFH



 


 651
CTGTEEHIGD CLMTALGAPT CSEGQVASVI CSGNQSQTLL PCSSLSPVQT



 


 701
TSSTIPKESE VPCIASGQLR LVGGGGRCAG RVEVYHEGSW GTVCDDNWDM



 


 751
TDANVVCKQL DCGVAINATG SAYFGEGAGA IWLDEVICTG KESHIWQCHS



 


 801
HGWGRHNCRH KEDAGVICSE FMSLRLTNEA HKENCTGRLE VFYNGTWGSI



 


 851
GSSNMSPTTV GVVCRQLGCA DNGTVKPIPS DKTPSRPMWV DRVQCPKGVD



 


 901
TLWQCPSSPW KQRQASPSSQ ESWIICDNKI RLQEGHTDCS GRVEIWHKGS



 


 951
WGTVCDDSWD LNDAKVVCKQ LGCGQAVKAL KEAAFGPGTG PIWLNEIKCR



 


1001
GNESSLWDCP AKPWSHSDCG HKEDASIQCL PKMTSESHHG TGHPTLTALL



 


1051
VCGAILLVLL IVFLLWTLKR RQIQRLTVSS RGEVLIHQVQ YQEMDSKADD



 


1101
LDLLKSSGVI QRHTEKENDN L



 









gctttggaat gggtggacac agaatggttc ttcttggagg tgctggatct cctggttgta
  60
ID NO: 25


 


aaaggtttgt ccatctaggt ttctttgttg tggctgtgag ctcacttctc agtgcctctg
 120



 


ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggcgggt ggtgaaaaca
 180



 


actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg
 240



 


gctggagcat gaatgaagtg tccgtggttt gccagcagct gggatgccca acttctatta
 300



 


aagcccttgg atgggctaac tccagcgccg gctctggata tatctggatg gacaaagttt
 360



 


cttgtacagg gaatgagtca gctctttggg actgcaaaca tgatgggtgg ggaaagcata
 420



 


actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga
 480



 


gactggtgaa cagtgcgggc caccgatgct taggaagagt agaaataaag ttccagggaa
 540



 


agtgggggac ggtgtgtgac gacaacttca gcaaagatca cgcttctgtg atttgtaaac
 600



 


agcttggatg tggaagtgcc attagtttct ctggctcagc taaattggga gctggttctg
 660



 


gaccaatctg gctcgatgac ctggcatgca atggaaatga gtcagctctc tgggactgca
 720



 


aacaccgggg atggggcaag cataactgtg accatgctga ggatgtcggt gtgatttgct
 780



 


tagagggagc agatctgagc ctgagactag tggatggagt gtccagatgt tcaggaagat
 840



 


tggaagtgag attccaagga gaatggggga ccgtgtgtga tgataactgg gatctccggg
 900



 


atgcttctgt ggtgtgcaag caactgggat gtccaactgc catcagtgcc attggtcgag
 960



 


ttaatgccag tgagggatct ggacagattt ggcttgacaa catttcatgc gaaggaaatg
1020



 


aggcaactct ttgggagtgt aaacaccaag agtggggaaa gcattactgt catcatagag
1080



 


aagacgctgg cgtgacatgt tctgatggag cagatctgga acttagactt gtaggtggag
1140



 


gcagtcgctg tgctggcatt gtggaggtgg agattcagaa gctgactggg aagatgtgta
1200



 


gccgaggctg gacactggca gatgcggatg tggtttgcag acagcttgga tgtggatctg
1260



 


cgcttcaaac ccaggctaag atctactcta aaactggggc aacaaatacg tggctctttc
1320



 


ctggatcttg taatggaaat gaaactactt tttggcaatg caaaaactgg cagtggggcg
1380



 


gcctttcctg tgataatttc gaagaagcca aagttacctg ctcaggccac agggaaccca
1440



 


gactggttgg aggagaaatc ccatgctctg gtcgtgtgga aatgaaacac ggagacgtgt
1500



 


ggggctccgt ctgtgatttt gacttgtctc tggaagctgc cagtgtggtg tgcagggaat
1560



 


tacaatgtgg aacagtcgtc tctatcctag ggggagcaca ttttggagaa ggaagtggac
1620



 


agatctgggg tgaagaattc cagtgtagtg gggatgagtc ccatctttca ctatgctcag
1680



 


tggcgccccc gctagacaga acttgtaccc acagcaggga tgtcagcgta gtctgctcac
1740



 


gatacataga tattcgtctg gcaggcggcg agtcctcctg tgagggaaga gtggagctca
1800



 


agacactcgg agcctggggt cccctctgca gttctcattg ggacatggaa gatgctcatg
1860



 


tcttatgtca gcagctgaag tgtggggttg cccaatctat tccagaagga gcacattttg
1920



 


ggaaaggagc tggtcaggtc tggagtcaca tgttccactg cactggaact gaggaacata
1980



 


taggagattg cctcatgact gctctgggtg cgccgacgtg ttccgaagga caggtggcct
2040



 


ctgtcatctg ctcaggaaac caatcccaga cactattgcc atgtagttca ttgtctccag
2100



 


tccaaacaac aagctctaca attccaaagg agagtgaagt tccctgcata gcaagtggcc
2160



 


agcttcgctt ggtaggtgga ggtggtcgct gcgctggaag agtggaggtc taccacgagg
2220



 


gctcttgggg caccgtctgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca
2280



 


agcagctgga ctgtggcgtg gcaattaacg ccactggctc tgcttacttc ggggaaggag
2340



 


caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggcagt
2400



 


gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct
2460



 


gctccgagtt catgtctctg aggctgacca acgaagccca caaagaaaac tgcacaggtc
2520



 


gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtctccaa
2580



 


ccactgtggg ggtggtgtgc cgtcagctgg gctgtgcaga caacgggact gtgaaaccca
2640



 


taccttcaga caagacacca tccaggccca tgtgggtaga tcgtgtgcag tgtccaaaag
2700



 


gagttgacac tttgtggcag tgcccctcgt caccttggaa acagagacag gccagcccct
2760



 


cctcccagga gtcctggatc atctgtgaca acaaaataag actccaggaa gggcatacag
2820



 


actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact
2880



 


cctgggatct taatgatgct aaggttgtat gtaagcagtt gggctgtggc caagctgtga
2940



 


aggcactaaa agaagcagca tttggtccag gaactgggcc catatggctc aatgaaatta
3000



 


agtgtagagg gaatgagtct tccctgtggg attgtcctgc caaaccgtgg agtcacagcg
3060



 


actgtgggca caaagaagat gcttccatcc agtgcctccc caaaatgact tcagaatcac
3120



 


atcatggcac aggtcacccc accctcacgg cactcttggt ttgtggagcc attctattgg
3180



 


tcctcctcat tgtcttcctc ctgtggactc tgaagcgacg acagattcag cgacttacag
3240



 


tttcctcaag aggagaggtc ttgatacatc aagttcagta ccaagagatg gattcaaagg
3300



 


cggatgatct ggacttgctg aaatcctcgg aaaattccaa caattcatat gattttaatg
3360



 


atgatggact gacatctttg tctaaatatc ttcctatttc tggaattaaa aaggggtcat
3420



 


tcagaggcac actgagaagg aaaatgataa tttataatcc actgaggttg gagtttaaga
3480



 


agccttgaca ggacagccag ctaaatggaa caagagccca ggcaacgcac ggatgaccac
3540



 


agctgcatct tcatgcagtc ctttgtttcc tggaactctg ctgaacctgc aaaaaccata
3600



 


tttgtgaatg tgaccactta atagagatgg gagactttt
3639



 









   1
M  G  G  H  R  M  V  L  L  G  G  A  G  S  P  G  C  K  R  F
ID NO: 26


   1
atgggtggacacagaatggttcttcttggaggtgctggatctcctggttgtaaaaggttt
and 27


 


  21
V  H  L  G  F  F  V  V  A  V  S  S  L  L  S  A  S  A  V  T



  61
gtccatctaggtttctttgttgtggctgtgagctcacttctcagtgcctctgctgtcact



 


  41
N  A  P  G  E  M  K  K  E  L  R  L  A  G  G  E  N  N  C  S



 121
aacgctcctggagaaatgaagaaggaactgagactggcgggtggtgaaaacaactgtagt



 


  61
G  R  V  E  L  K  I  H  D  K  W  G  T  V  C  S  N  G  W  S



 181
gggagagtggaacttaagatccatgacaagtggggcacagtgtgcagtaacggctggagc



 


  81
M  N  E  V  S  V  V  C  Q  Q  L  G  C  P  T  S  I  K  A  L



 241
atgaatgaagtgtccgtggtttgccagcagctgggatgcccaacttctattaaagccctt



 


 101
G  W  A  N  S  S  A  G  S  G  Y  I  W  M  D  K  V  S  C  T



 301
ggatgggctaactccagcgccggctctggatatatctggatggacaaagtttcttgtaca



 


 121
G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C  T



 361
gggaatgagtcagctctttgggactgcaaacatgatgggtggggaaagcataactgtacc



 


 141
H  E  K  D  A  G  V  T  C  S  D  G  S  N  L  E  M  R  L  V



 421
catgaaaaagatgctggagtgacctgctcagatggatctaatttggagatgagactggtg



 


 161
N  S  A  G  H  R  C  L  G  R  V  E  I  K  F  Q  G  K  W  G



 481
aacagtgcgggccaccgatgcttaggaagagtagaaataaagttccagggaaagtggggg



 


 181
T  V  C  D  D  N  F  S  K  D  H  A  S  V  I  C  K  Q  L  G



 541
acggtgtgtgacgacaacttcagcaaagatcacgcttctgtgatttgtaaacagcttgga



 


 201
C  G  S  A  I  S  F  S  G  S  A  K  L  G  A  G  S  G  P  I



 601
tgtggaagtgccattagtttctctggctcagctaaattgggagctggttctggaccaatc



 


 221
W  L  D  D  L  A  C  N  G  N  E  S  A  L  W  D  C  K  H  R



 661
tggctcgatgacctggcatgcaatggaaatgagtcagctctctgggactgcaaacaccgg



 


 241
G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  E  G



 721
ggatggggcaagcataactgtgaccatgctgaggatgtcggtgtgatttgcttagaggga



 


 261
A  D  L  S  L  R  L  V  D  G  V  S  R  C  S  G  R  L  E  V



 781
gcagatctgagcctgagactagtggatggagtgtccagatgttcaggaagattggaagtg



 


 281
R  F  Q  G  E  W  G  T  V  C  D  D  N  W  D  L  R  D  A  S



 841
agattccaaggagaatgggggaccgtgtgtgatgataactgggatctccgggatgcttct



 


 301
V  V  C  K  Q  L  G  C  P  T  A  I  S  A  I  G  R  V  N  A



 901
gtggtgtgcaagcaactgggatgtccaactgccatcagtgccattggtcgagttaatgcc



 


 321
S  E  G  S  G  Q  I  W  L  D  N  I  S  C  E  G  H  E  A  T



 961
agtgagggatctggacagatttggcttgacaacatttcatgcgaaggacatgaggcaact



 


 341
L  W  E  C  K  H  Q  E  W  G  K  H  Y  C  H  H  R  E  D  A



1021
ctttgggagtgtaaacaccaagagtggggaaagcattactgtcatcatagagaagacgct



 


 361
G  V  T  C  S  D  G  A  D  L  E  L  R  L  V  G  G  G  S  R



1081
ggcgtgacatgttctgatggagcagatctggaacttagacttgtaggtggaggcagtcgc



 


 381
C  A  G  I  V  E  V  E  I  Q  K  L  T  G  K  M  C  S  R  G



1141
tgtgctggcattgtggaggtggagattcagaagctgactgggaagatgtgtagccgaggc



 


 401
W  T  L  A  D  A  D  V  V  C  R  Q  L  G  C  G  S  A  L  Q



1201
tggacactggcagatgcggatgtggtttgcagacagcttggatgtggatctgcgcttcaa



 


 421
T  Q  A  K  I  Y  S  K  T  G  A  T  N  T  W  L  F  P  G  S



1261
acccaggctaagatctactctaaaactggggcaacaaatacgtggctctttcctggatct



 


 441
C  N  G  N  E  T  T  F  W  Q  C  K  N  W  Q  W  G  G  L  S



1321
tgtaatggaaatgaaactactttttggcaatgcaaaaactggcagtggggcggcctttcc



 


 461
C  D  N  F  E  E  A  K  V  T  C  S  G  H  R  E  P  R  L  V



1381
tgtgataatttcgaagaagccaaagttacctgctcaggccacagggaacccagactggtt



 


 481
G  G  E  I  P  C  S  G  R  V  E  M  K  H  G  D  V  W  G  S



1441
ggaggagaaatcccatgctctggtcgtgtggaaatgaaacacggagacgtgtggggctcc



 


 501
V  C  D  F  D  L  S  L  E  A  A  S  V  V  C  R  E  L  Q  C



1501
gtctgtgattttgacttgtctctggaagctgccagtgtggtgtgcagggaattacaatgt



 


 521
G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  S  G  Q  I  W



1561
ggaacagtcgtctctatcctagggggagcacattttggagaaggaagtggacagatctgg



 


 541
G  E  E  F  Q  C  S  G  D  E  S  H  L  S  L  C  S  V  A  P



1621
ggtgaagaattccagtgtagtggggatgagtcccatctttcactatgctcagtggcgccc



 


 561
P  L  D  R  T  C  T  H  S  R  D  V  S  V  V  C  S  R  Y  I



1681
ccgctagacagaacttgtacccacagcagggatgtcagcgtagtctgctcacgatacata



 


 581
D  I  R  L  A  G  G  E  S  S  C  E  G  R  V  E  L  K  T  L



1741
gatattcgtctggcaggcggcgagtcctcctgtgagggaagagtggagctcaagacactc



 


 601
G  A  W  G  P  L  C  S  S  H  W  D  M  E  D  A  H  V  L  C



1801
ggagcctggggtcccctctgcagttctcattgggacatggaagatgctcatgtcttatgt



 


 621
Q  Q  L  K  C  G  V  A  Q  S  I  P  E  G  A  H  F  G  K  G



1861
cagcagctgaagtgtggggttgcccaatctattccagaaggagcacattttgggaaagga



 


 641
A  G  Q  V  W  S  H  M  F  H  C  T  G  T  E  E  H  I  G  D



1921
gctggtcaggtctggagtcacatgttccactgcactggaactgaggaacatataggagat



 


 661
C  L  M  T  A  L  G  A  P  T  C  S  E  G  Q  V  A  S  V  I



1981
tgcctcatgactgctctgggtgcgccgacgtgttccgaaggacaggtggcctctgtcatc



 


 681
C  S  G  N  Q  S  Q  T  L  L  P  C  S  S  L  S  P  V  Q  T



2041
tgctcaggaaaccaatcccagacactattgccatgtagttcattgtctccagtccaaaca



 


 701
T  S  S  T  I  P  K  E  S  E  V  P  C  I  A  S  G  Q  L  R



2101
acaagctctacaattccaaaggagagtgaagttccctgcatagcaagtggccagcttcgc



 


 721
L  V  G  G  G  G  R  C  A  G  R  V  E  V  Y  H  E  G  S  W



2161
ttggtaggtggaggtggtcgctgcgctggaagagtggaggtctaccacgagggctcttgg



 


 741
G  T  V  C  D  D  N  W  D  M  T  D  A  N  V  V  C  K  Q  L



2221
ggcaccgtctgtgatgacaattgggatatgactgatgccaatgtggtgtgcaagcagctg



 


 761
D  C  G  V  A  I  N  A  T  G  S  A  Y  F  G  E  G  A  G  A



2281
gactgtggcgtggcaattaacgccactggctctgcttacttcggggaaggagcaggagct



 


 781
I  W  L  D  E  V  I  C  T  G  K  E  S  H  I  W  Q  C  H  S



2341
atctggctagacgaagtcatctgcactgggaaagagtctcatatttggcagtgccattca



 


 801
H  G  W  G  R  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
catggctggggacgccataactgcaggcacaaagaagatgcaggtgttatctgctccgag



 


 821
F  M  S  L  R  L  T  N  E  A  H  K  E  N  C  T  G  R  L  E



2461
ttcatgtctctgaggctgaccaacgaagcccacaaagaaaactgcacaggtcgccttgaa



 


 841
V  F  Y  N  G  T  W  G  S  I  G  S  S  N  M  S  P  T  T  V



2521
gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctccaaccactgtg



 


 861
G  V  V  C  R  Q  L  G  C  A  D  N  G  T  V  K  P  I  P  S



2581
ggggtggtgtgccgtcagctgggctgtgcagacaacgggactgtgaaacccataccttca



 


 881
D  K  T  P  S  R  P  M  W  V  D  R  V  Q  C  P  K  G  V  D



2641
gacaagacaccatccaggcccatgtgggtagatcgtgtgcagtgtccaaaaggagttgac



 


 901
T  L  W  Q  C  P  S  S  P  W  K  Q  R  Q  A  S  P  9  S  Q



2701
actttgtggcagtgcccctcgtcaccttggaaacagagacaggccagcccctcctcccag



 


 921
E  S  W  I  I  C  D  N  K  I  R  L  Q  E  G  H  T  D  C  S



2761
gagtcctggatcatctgtgacaacaaaataagactccaggaagggcatacagactgttct



 


 941
G  R  V  E  I  W  H  K  G  S  W  G  T  V  C  D  D  S  W  D



2821
ggacgtgtggagatctggcacaaaggttcctggggaacagtgtgtgatgactcctgggat



 


 961
L  N  D  A  K  V  V  C  K  Q  L  G  C  G  Q  A  V  K  A  L



2881
cttaatgatgctaaggttgtatgtaagcagttgggctgtggccaagctgtgaaggcacta



 


 981
K  E  A  A  F  G  P  G  T  G  P  I  W  L  N  E  I  K  C  R



2941
aaagaagcagcatttggtccaggaactgggcccatatggctcaatgaaattaagtgtaga



 


1001
G  N  E  S  S  L  W  D  C  P  A  K  P  W  S  H  S  D  C  G



3001
gggaatgagtcttccctgtgggattgtcctgccaaaccgtggagtcacagcgactgtggg



 


1021
H  K  E  D  A  S  I  Q  C  L  P  K  M  T  S  E  S  H  H  G



3061
cacaaagaagatgcttccatccagtgcctccccaaaatgacttcagaatcacatcatggc



 


1041
T  G  H  P  T  L  T  A  L  L  V  C  G  A  I  L  L  V  L  L



3121
acaggtcaccccaccctcacggcactcttggtttgtggagccattctattggtcctcctc



 


1061
I  V  F  L  L  W  T  L  K  R  R  Q  I  Q  R  L  T  V  S  S



3181
attgtcttcctcctgtggactctgaagcgacgacagattcagcgacttacagtttcctca



 


1081
R  G  E  V  L  I  H  Q  V  Q  Y  Q  E  M  D  S  K  A  D  D



3241
agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggcggatgat



 


1101
L  D  L  L  K  S  S  E  N  S  N  N  S  Y  D  F  N  D  D  G



3301
ctggacttgctgaaatcctcggaaaattccaacaattcatatgattttaatgatgatgga



 


1121
L  T  S  L  S  K  Y  L  P  I  S  G  I  K  K  G  S  F  R  G



3361
ctgacatctttgtctaaatatcttcctatttctggaattaaaaaggggtcattcagaggc



 


1141
T  L  R  R  K  M  I  I  Y  N  P  L  R  L  E  F  K  K  P



3421
acactgagaaggaaaatgataatttataatccactgaggttggagtttaagaagcct



 


   1
MGGHRMVLLG GAGSPGCKRF VHLGFFVVAV SSLLSASAVT NAPGEMKKEL
ID NO: 27


 


  51
RLAGGENNCS GRVELKIHDK WGTVCSNGWS MNEVSVVCQQ LGCPTSIKAL



 


 101
GWANSSAGSG YIWMDKVSCT GNESALWDCK HDGWGKHNCT HEKDAGVTCS



 


 151
DGSNLEMRLV NSAGHRCLGR VEIKFQGKWG TVCDDNFSKD HASVICKQLG



 


 201
CGSAISFSGS AKLGAGSGPI WLDDLACNGN ESALWDCKHR GWGKHNCDHA



 


 251
EDVGVICLEG ADLSLRLVDG VSRCSGRLEV RFQGEWGTVC DDNWDLRDAS



 


 301
VVCKQLGCPT AISAIGRVNA SEGSGQIWLD NISCEGHEAT LWECKHQEWG



 


 351
KHYCHHREDA GVTCSDGADL ELRLVGGGSR CAGIVEVEIQ KLTGKMCSRG



 


 401
WTLADADVVC RQLGCGSALQ TQAKIYSKTG ATNTWLFPGS CNGNETTFWQ



 


 451
CKNWQWGGLS CDNFEEAKVT CSGHREPRLV GGEIPCSGRV EMKHGDVWGS



 


 501
VCDFDLSLEA ASVVCRELQC GTVVSILGGA HFGEGSGQIW GEEFQCSGDE



 


 551
SHLSLCSVAP PLDRTCTHSR DVSVVCSRYI DIRLAGGESS CEGRVELKTL



 


 601
GAWGPLCSSH WDMEDAHVLC QQLKCGVAQS IPEGAREGKG AGQVWSHMFH



 


 651
CTGTEEHIGD CLMTALGAPT CSEGQVASVI CSGNQSQTLL PCSSLSPVQT



 


 701
TSSTIPKESE VPCIASGQLR LVGGGGRCAG RVEVYHEGSW GTVCDDNWDM



 


 751
TDANVVCKQL DCGVAINATG SAYFGEGAGA IWLDEVICTG KESHIWQCHS



 


 801
HGWGRHNCRH KEDAGVICSE FMSLRLTNEA HKENCTGRLE VFYNGTWGSI



 


 851
GSSNMSPTTV GVVCRQLGCA DNGTVKPIPS DKTPSRPMWV DRVQCPKGVD



 


 901
TLWQCPSSPW KQRQASPSSQ ESWIICDNKI RLQEGHTDCS GRVEIWHKGS



 


 951
WGTVCDDSWD LNDAKVVCKQ LGCGQAVKAL KEAAFGPGTG PIWLNEIKCR



 


1001
GNESSLWDCP AKPWSHSDCG HKEDASIQCL PKMTSESHHG TGHPTLTALL



 


1051
VCGAILLVLL IVFLLWTLKR RQIQRLTVSS RGEVLIHQVQ YQEMDSKADD



 


1101
LDLLKSSENS NNSYDFNDDG LTSLSKYLPI SGIKKGSFRG TLRRKMIIYN



 


1151
PLRLEFKKP









Example 8
Cloning and Characterization of MARC-145 CD163
A forward primer 5′ simianCD163 (SEQ ID NO: 28) (5′-CACCGGAATGAGCAAACTCAGAATGG-3′ based on human CD163) and a reverse primer HuCD163-3′Kpn (SEQ ID NO:29) (5′-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were used to amplify CD163 cDNA from MARC-145 African Green Monkey kidney cells. Total cellular RNA was prepared from MARC-145 cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer's instruction. Several clones containing large inserts were analyzed. Clone #25 was designated “pCDNA3.1D-MARC-CD163v2”. This novel CD163 cDNA from MARC-145 cells is 1116 amino acids in length. When compared to the sequences in GenBank database, the MARC-145 CD163 amino acid sequence is 96.3% identical to human CD163 (Genbank Z22968), 84.7% identical to pig CD163 (Genbank AJ311716), and 73.9% identical to mouse CD163 (Genbank AF274883).











SEQUENCE
ID NO







atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat   60
SEQ ID


tttgtcaact tgagtccctt cactattgct gtggtcttac ttctccgtgc ctgttttgtc  120
NO: 30


accagttctc ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt  180



agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg  240



agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcc  300



actggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttcttgt  360



cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac  420



tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg  480



ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg  540



ggaacagtgt gtgatgataa cttcaacatc aatcatgcat ctgtggtttg taaacaactt  600



gaatgtggaa gtgctgtcag tttctctggt tcagctaatt ttggagaagg ctctggacca  660



atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat  720



caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag  780



ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa  840



gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct  900



gctgtggcat gcaagcaact gggatgtcca actgctatca ccgccattgg tcgagttaac  960



gccagtgagg gatttggaca catctggctt gacagtgttt cttgccaggg acatgaacct 1020



gcggtctggc aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080



gctggcgtaa catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140



cgctgtgctg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200



ggctggggac tgaaagaagc tgatgtggtt tgcaggcagc tgggatgtgg atctgcactc 1260



aaaacatcct atcaagtata ctccaaaatc caggcaacaa acatgtggct gtttctaagt 1320



agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380



acctgtgatc actatgaaga agccaaaatt acctgctcag cccacaggga acccagactg 1440



gttggaggag acattccctg ttctggacgc gttgaagtga agcatggtga cacatggggc 1500



tccgtctgtg attcggattt ctctctggaa gctgccagcg ttctatgcag ggaattacag 1560



tgtggcacag tcgtctctat cctgggggga gctcactttg gagagggaaa tggacagatc 1620



tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680



ccccgcccag aaggaacttg tagccacagc agggatgttg gagtagtctg ctcaagatac 1740



acagaaattc gcttggtgaa tggcaagacc ccatgtgagg gcagagtgga gctcaaaacg 1800



cttaatgcct ggggatccct ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860



tgccaacaac ttaaatgtgg agttgccctt tctaccccag gaggagcaca ttttggaaaa 1920



ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980



gattgtcctg taactgctct gggtgcttca ctatgtcctt cagggcaagt ggcctctgta 2040



atttgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcatc tctgggccca 2100



acaaggccta ccattccaga agaaagtgct gtggcctgca tagagagtgg tcaacttcgc 2160



ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggctcctgg 2220



ggcaccatct gtgatgacag ctgggacctg agcgatgccc acgtggtgtg cagacagctg 2280



ggctgtggag aggccattaa tgccactggt tctgctcatt ttggagaagg aacagggccc 2340



atctggctgg atgagatgaa atgcaatgga aaagaatccc gcatttggca gtgccattca 2400



catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460



ttcatgtctc tgagactgac cagtgaagcc agcagagagg cctgtgcagg gcgtctagaa 2520



gttttttaca acggagcttg gggcagtgtt ggcaggagta acatgtctga aaccactgtg 2580



ggtgtggtgt gcaggcagct gggctgtgca gacaaaggga aaatcaaccc tgcatcttta 2640



gacaaggcca tgtccattcc catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700



acgctgtggc agtgcccatc atctccatgg gagaagagac tggccaggcc ctcggaggag 2760



acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820



cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880



aacgatgctc aggtggtgtg tcaacaactt ggctgtggtc cagctttgaa agcattcaaa 2940



gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000



aatgagtctt ccttgtggga ttgtcctgcc agacgctggg gccacagtga gtgtggacac 3060



aaggaagacg ctgcagtgaa ttgcacagat atttcaacga acaaaacccc acaaaaagcc 3120



acaacaggtc agtcatccct tattgcagtc ggaatccttg gagttgttct cttggtcatt 3180



ttcgtcgcat tattcttgac tcaaaagcga agacagagac agcggcttac agtttcctca 3240



agaggagaga acttagtcca ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300



gatgatctgg acctaatgaa ttcctcagga ggccattctg aggcacactg aaaaggaaaa 3360



tgggaattta taacccagtg agccttgaag ataccttgat gaagacctgg acta       3414



 


   1 M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H
SEQ ID NO:


   1 atgagcaaactcagaatggtgctacttgaagactctggatctgctgacgtcagaagacat
31 and 32


 


  21 F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



  61 tttgtcaacttgagtcccttcactattgctgtggtcttacttctccgtgcctgttttgtc



 


  41 T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



 121 accagttctcttggaggaacaaccaaggagctgaggctagtggatggtgaaaacaagtgt



 


  61 S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



 181 agtgggagagtggaagtgaaaatccaggaggagtggggaacggtgtgtaataatggctgg



 


  81 S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



 241 agcatggaagcagtctctgtgatttgtaaccagctgggatgtccaactgctatcaaagcc



 


 101 T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



 301 actggatgggctaattccagtgcaggttctggacgcatttggatggatcatgtttcttgt



 


 121 R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



 361 cgtgggaatgagtcagctctttgggactgcaaacatgatggatggggaaagcatagtaac



 


 141 C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



 421 tgtactcaccaacaagatgctggagtgacttgctcagatggatccgatttggaaatgagg



 


 161 L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W



 481 ctgacgaatggagggaatatgtgttctggaagaatagagatcaaattccaaggacagtgg



 


 181 G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L



 541 ggaacagtgtgtgatgataacttcaacatcaatcatgcatctgtggtttgtaaacaactt



 


 201 E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P



 601 gaatgtggaagtgctgtcagtttctctggttcagctaattttggagaaggctctggacca



 


 221 I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



 661 atctggtttgatgatcttatatgcaacggaaatgagtcagctctctggaactgcaaacat



 


 241 Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



 721 caaggatggggaaagcataactgtgatcatgctgaggatgctggagtgatttgctcaaag



 


 261 G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



 781 ggagcagatctgagcctgagactggtagatggagtcactgaatgttcaggaagattagaa



 


 281 V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



 841 gtgagattccaaggagaatgggggacaatatgtgatgacggctgggacagtcatgatgct



 


 301 A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



 901 gctgtggcatgcaagcaactgggatgtccaactgctatcaccgccattggtcgagttaac



 


 321 A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



 961 gccagtgagggatttggacacatctggcttgacagtgtttcttgccagggacatgaacct



 


 341 A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  G  N  H  N  E  D



1021 gcggtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat



 


 361 A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S



1081 gctggcgtaacatgttctgatggatcagatctggagctaagacttagaggtggaggcagc



 


 381 R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141 cgctgtgctgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga



 


 401 G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201 ggctggggactgaaagaagctgatgtggtttgcaggcagctgggatgtggatctgcactc



 


 421 K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261 aaaacatcctatcaagtatactccaaaatccaggcaacaaacatgtggctgtttctaagt



 


 441 S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321 agctgtaacggaaatgaaacttctctttgggactgcaagaactggcaatggggtggactt



 


 461 T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381 acctgtgatcactatgaagaagccaaaattacctgctcagcccacagggaacccagactg



 


 481 V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441 gttggaggagacattccctgttctggacgcgttgaagtgaagcatggtgacacatggggc



 


 501 S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501 tccgtctgtgattcggatttctctctggaagctgccagcgttctatgcagggaattacag



 


 521 C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561 tgtggcacagtcgtctctatcctggggggagctcactttggagagggaaatggacagatc



 


 541 W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621 tgggctgaagaattccagtgtgagggacatgagtcccatctttcactctgcccagtagca



 


 561 P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681 ccccgcccagaaggaacttgtagccacagcagggatgttggagtagtctgctcaagatac



 


 581 T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741 acagaaattcgcttggtgaatggcaagaccccatgtgagggcagagtggagctcaaaacg



 


 601 L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801 cttaatgcctggggatccctctgcaactctcactgggacatagaagatgcccacgttctt



 


 621 C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861 tgccaacaacttaaatgtggagttgccctttctaccccaggaggagcacattttggaaaa



 


 641 G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921 ggaaatggtcaggtctggaggcatatgtttcactgcactgggactgagcagcacatggga



 


 661 D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981 gattgtcctgtaactgctctgggtgcttcactatgtccttcagggcaagtggcctctgta



 


 681 I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041 atttgctcaggaaaccagtcccaaacactgtcctcgtgcaattcatcatctctgggccca



 


 701 T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101 acaaggcctaccattccagaagaaagtgctgtggcctgcatagagagtggtcaacttcgc



 


 721 L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161 ttggtaaatggaggaggtcgctgtgctgggagagtagagatttatcatgagggctcctgg



 


 741 G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221 ggcaccatctgtgatgacagctgggacctgagcgatgcccacgtggtgtgcagacagctg



 


 761 G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281 ggctgtggagaggccattaatgccactggttctgctcattttggagaaggaacagggccc



 


 781 I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341 atctggctggatgagatgaaatgcaatggaaaagaatcccgcatttggcagtgccattca



 


 801 H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401 catggctgggggcagcaaaactgcaggcacaaggaggatgcaggagttatctgctcagag



 


 821 F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461 ttcatgtctctgagactgaccagtgaagccagcagagaggcctgtgcagggcgtctagaa



 


 841 V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V



2521 gttttttacaacggagcttggggcagtgttggcaggagtaacatgtctgaaaccactgtg



 


 861 G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  P  A  S  L



2581 ggtgtggtgtgcaggcagctgggctgtgcagacaaagggaaaatcaaccctgcatcttta



 


 881 D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641 gacaaggccatgtccattcccatgtgggtggacaatgttcagtgtccaaaaggacctgac



 


 901 T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701 acgctgtggcagtgcccatcatctccatgggagaagagactggccaggccctcggaggag



 


 921 T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761 acctggatcacatgtgacaacaagatgagactacaagaaggacccacttcctgttctgga



 


 941 R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821 cgtgtggagatctggcacggaggttcctgggggacagtgtgtgatgactcctgggacttg



 


 961 N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881 aacgatgctcaggtggtgtgtcaacaacttggctgtggtccagctttgaaagcattcaaa



 


 981 E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941 gaagcagagtttggtcaggggactggacccatatggctcaatgaagtgaagtgcaaaggg



 


1001 N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001 aatgagtcttccttgtgggattgtcctgccagacgctggggccacagtgagtgtggacac



 


1021 K  E  D  A  A  V  N  C  T  D  I  S  T  N  K  T  P  Q  K  A



3061 aaggaagacgctgcagtgaattgcacagatatttcaacgaacaaaaccccacaaaaagcc



 


1041 T  T  G  Q  S  S  L  I  A  V  G  I  L  G  V  V  L  L  V  I



3121 acaacaggtcagtcatcccttattgcagtcggaatccttggagttgttctcttggtcatt



 


1061 F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V  S  S



3181 ttcgtcgcattattcttgactcaaaagcgaagacagagacagcggcttacagtttcctca



 


1081 R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  N  A



3241 agaggagagaacttagtccaccaaattcaataccgggagatgaattcttgcctgaatgca



 


1101 D  D  L  D  L  M  N  S  S  G  G  H  S  E  A  H



3301 gatgatctggacctaatgaattcctcaggaggccattctgaggcacac



 


   1 MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE
SEQ ID


  51 LRLVDGENKC SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA
NO: 32


 101 TGWANSSAGS GRIWNDHVSC RGNESALWDC KHDGWGKHSN CTHQQDAGVT



 151 CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW CTVCDDNFNI NHASVVCKQL



 201 ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH QGWGKHNCDH



 251 AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



 301 AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW



 351 GKHYCNHNED AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLCKVCDR



 401 GWGLKEADVV CRQLGCGSAL KTSYQVYSKI QATNMWLFLS SCNGNETSLW



 451 DCKNWQWGGL TCDHYEEAKI TCSAHREPRL VGGDIPCSGR VEVKHGDTWG



 501 SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI WAEEFQCECH



 551 ESHLSLCPVA PRPECTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



 601 LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF



 651 HCTGTEQHMG DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP



 701 TRPTIPEESA VACIESGQLR LVNGGGRCAG RVEIYHEGSW GTICDDSWDL



 751 SDAHVVCRQL GCGEAINATG SAHFGEGTGP IWLDEMKCNG KESRIWQCHS



 801 HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE VFYNGAWGSV



 851 GRSNMSETTV GVVCRQLGCA DKGKINPASL DKAMSIPMWV DNVQCPKGPD



 901 TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW



 951 GTVCDDSWDL NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG



1001 NESSLWDCPA RRWGHSECGH KEDAAVNCTD ISTNKTPQKA TTGQSSLIAV



1051 GILGVVLLVI FVALFLTQKR RQRQRLTVSS RGENLVHQIQ YREMNSCLNA



1101 DDLDLMNSSG GHSEAH









Example 9
Cloning and Characterization of Simian CD163 from Vero Cells
A forward primer 5′ simianCD163 (SEQ ID NO: 28) (5′-CACCGGAATGAGCAAACTCAGAATGG-3′ based on human CD163) and a reverse primer HuCD163-3′Kpn (SEQ ID NO:29) (5′-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3′) were used to amplify CD163 cDNA from Vero cells. Total cellular RNA was prepared from Vero cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer's instruction. Eight clones containing large inserts were sequenced, and six discreet splicing patterns were found. These patterns are depicted graphically in FIG. 17.
The six splicing variants differ in the presence or absence of three exons, designated E6, E105, and E83. Omissions of E6 or E105 do not change the reading frame, whereas omission of E83 does. Patterns similar to v2 and/or v3 were also seen in porcine, murine, human, and MARC-145 monkey cells. Patterns v4 and v5 lack the 105-nucleotide exon that encodes the hydrophobic transmembrane region. These cDNAs were unable to render BHK cells permissive to PRRSV infection in a transient transfection assay, probably because the CD163 is secreted rather than remaining membrane bound. Although CD163 molecules lacking a transmembrane region appear to be non-functional as cellular permissivity factors, it is possible that they may have utility either in direct virus neutralization (similar to neutralizing antibodies), or as an immunogen for the induction of anti-CD163 antibodies which would block viral infection in the host animal.
The longest splice variant, v7, contains all three of the exons E6, E105, and E83. This novel CD163 cDNA from Vero cells encodes a polypeptide that is 1153 amino acids in length. When compared to the sequences in Genbank database, the Vero CD163v7 amino acid sequence is 95.4% identical to human CD163 (Genbank Z22968), 83.7% identical to pig CD163 (Genbank AJ311716), and 72.1% identical to mouse CD163 (Genbank AF274883). The nucleotide and amino acid sequences of the six splice variants found in Vero cells are provided below (SEQ ID NOS:33-44).











SEQUENCE
ID NO

















1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H
SEQ ID NO:


1
ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT
33 and 34


 


21
F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



61
TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC



 


41
T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



121
ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT



 


61
S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



181
AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



241
AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC



 


101
T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



301
ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



361
CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC



 


141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



421
TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG



 


161
L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W



481
CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG



 


181
G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L



541
GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT



 


201
E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P



601
GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA



 


221
I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



661
ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT



 


241
Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



721
CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG



 


261
G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



781
GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCATGAATGTTCAGGAAGATTAGAA



 


281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



841
GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT



 


301
A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



901
GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC



 


321
A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



961
GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT



 


341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT



 


361
A  G  V  T  C  S  D  G  S  G  L  E  L  R  L  R  G  G  G  S



1081
GCTGGCGTAACATGTTCTGATGGATCAGGTCTGGAGCTAAGACTTAGAGGTGGAGGCAGC



 


381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA



 


401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC



 


421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261
AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT



 


441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT



 


461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381
ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG



 


481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC



 


501
S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG



 


521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC



 


541
W  T  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA



 


561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC



 


581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG



 


601
L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT



 


621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861
TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA



 


641
G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA



 


661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981
GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA



 


681
I  C  S  G  N  Q  S  Q  T  L  S  S  R  N  S  S  S  L  G  P



2041
ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGCGCAATTCATCATCTCTGGGCCCA



 


701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC



 


721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161
TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG



 


741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG



 


761
G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC



 


781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCA



 


801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG



 


821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
TTCATGTCTCTGAGACTGACCAGTGAAGCCAGGAGAGAGGCCTGTGCAGGGCGTCTAGAA



 


841
V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V



2521
GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG



 


861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L



2581
GGTGTAGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA



 


881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC



 


901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701
ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG



 


921
T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761
ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA



 


941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG



 


961
N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA



 


981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  E  G



2941
GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCGAAGGG



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC



 


1021
K  E  D  A  A  V  N  C  T  D  I  S  T  R  K  T  P  Q  K  A



3061
AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC



 


1041
T  T  G  Q  S  S  L  I  A  V  G  I  L  I  V  V  L  L  A  I



3121
ACAACAGGTCAGTCATCCCTTATTGCAGTCGGAATCCTTGGAGTTGTTCTCTTGGCCATT



 


1061
F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V  S  S



3181
TTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTTTCCTCA



 


1081
R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  N  A



3241
AGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTGAATGCA



 


1101
D  D  L  D  L  M  N  S  S  G  G  H  S  E  A  H



3301
GATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCACAC



 


1
MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC
SEQ ID


61
SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC
NO: 34


121
RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW



181
GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH



241
QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



301
AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED



361
AGVTCSDGSG LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL



421
KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL



481
VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI



541
WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



601
LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG



661
DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSRNSSSLGP TRPTIPEESA VACIESGQLR



721
LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP



781
IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE



841
VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD



901
TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL



961
NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCEG NESSLWDCPA RRWGHSECGH



1021
KEDAAVNCTD ISTRKTPQKA TTGQSSLIAV GILGVVLLAI FVALFLTQKR RQRQRLTVSS



1081
RGENLVHQIQ YREMNSCLNA DDLDLMNSSG GHSEAH



 


1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H 
SEQ ID NO:


1
ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT
35 and 36


 


21
F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



61
TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC



 


41
T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



121
ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT



 


61
S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



181
AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



241
AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC



 


101
T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



301
ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



361
CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC



 


141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



421
TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG



 


161
L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W



481
CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG



 


181
G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L



541
GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT



 


201
E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P



601
GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA



 


221
I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



661
ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT



 


241
Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



721
CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG



 


261
G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



781
GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA



 


281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



841
GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT



 


301
A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



901
GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC



 


321
A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



961
GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT



 


341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT



 


361
A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S



1081
GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC



 


381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
CGCTGTGCTGGgACAGTTGAGGTgGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA



 


401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC



 


421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261
AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT



 


441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT



 


461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381
ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG



 


481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC



 


501
S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG



 


521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
TGTGGCACAGTCGTCTCTATCCTGGGGGAGGCTCACTTTGGAGAGGGAAATGGACAGATC



 


541
W  T  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA



 


561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC



 


581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG



 


601
L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT



 


621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861
TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA



 


641
G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA



 


661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981
GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA



 


681
I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041
ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA



 


701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC



 


721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161
TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG



 


741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG



 


761
G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC



 


781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCA



 


801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG



 


821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA



 


841
V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V



2521
GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG



 


861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L



2581
GGTGTAGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA



 


881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC



 


901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701
ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG



 


921
T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761
ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA



 


941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG



 


961
N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA



 


981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941
GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC



 


1021
K  E  D  A  A  V  N  C  T  D  I  S  T  R  K  T  P  Q  K  A



3061
AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC



 


1041
T  T  G  Q  S  S  L  I  A  V  G  I  L  G  V  V  L  L  A  I



3121
ACAACAGGTCAGTCATCCCTTATTGCAGTCGGAATCCTTGGAGTTGTTCTCTTGGCCATT



 


1061
F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V  S  S



3181
TTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTTTCCTCA



 


1081
R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L  N  A



3241
AGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTGAATGCA



 


1101
D  D  L  D  L  M  N  S  S  E  N  S  N  E  S  A  D  F  N  A



3301
GATGATCTGGACCTAATGAATTCCTCAGAAAATTCCAATGAGTCAGCTGATTTCAATGCT



 


1121
A  E  L  I  S  V  S  K  F  L  P  I  S  G  M  E  K  E  A  I



3361
GCTGAACTAATTTCTGTGTCTAAATTTCTTCCTATTTCTGGAATGGAAAAGGAGGCCATT



 


1141
L  R  H  T  E  K  E  N  G  N  L



3421
CTGAGGCACACTGAAAAGGAAAATGGGAATTTA



 


1
MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC
SEQ ID


61
SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC
NO: 36


121
RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW



181
GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH



241
QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



301
AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED



361
AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL



421
KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL



481
VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI



541
WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



601
LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG



661
DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR



721
LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP



781
IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE



841
VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD



901
TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL



961
NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH



1021
KEDAAVNCTD ISTRKTPQKA TTGQSSLIAV GILGVVLLAI FVALFLTQKR RQRQRLTVSS



1081
RGENLVHQIQ YREMNSCLNA DDLDLMNSSE NSNESADFNA AELISVSKFL PISGMEKEAI



1141
LRHTEKENGN L



 


1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H
SEQ ID NO:


1
ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT
37 and 38


 


21
F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



61
TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC



 


41
T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



121
ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT



 


61
S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



181
AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



241
AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC



 


101
T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



301
ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



361
CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC



 


141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



421
TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG



 


161
L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W



481
CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG



 


181
G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L



541
GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT



 


201
E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P



601
GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA



 


221
I  W  F  D  D  L  I  C  H  G  N  E  S  A  L  W  N  C  K  H



661
ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT



 


241
Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



721
CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG



 


261
G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



781
GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA



 


281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



841
GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT



 


301
A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



901
GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC



 


321
A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



961
GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT



 


341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT



 


361
A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  A  G  G  G  S



1081
GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC



 


381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA



 


401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC



 


421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261
AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT



 


441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT



 


461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381
ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG



 


481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC



 


501
S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG



 


521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC



 


541
W  T  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA



 


561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC



 


581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG



 


601
L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT



 


621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861
TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA



 


641
G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA



 


661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981
GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA



 


681
I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041
ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA



 


701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC



 


721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161
TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG



 


741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG



 


761
G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC



 


781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGCATTTGGCAGTGCCATTCA



 


801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG



 


821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA



 


841
V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V



2521
GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG



 


861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L



2581
GGTGTAGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA



 


881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC



 


901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701
ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG



 


921
T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761
ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA



 


941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG



 


961
N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA



 


981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941
GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC



 


1021
K  E  D  A  A  V  N  C  T  D  I  S  T  R  K  T  P  Q  K  A



3061
AAGGAAGACGCTGCGGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC



 


1041
T  T  V  S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N



3121
ACAACGGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAAT



 


1061
S  C  L  N  A  D  D  L  N  L  M  N  S  S  G  G  H  S  E  A



3181
TCTTGCCTGAATGCAGATGATCTGAACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCA



 


1081
H  *  K  G  K  W  E  F  I  T  Q



3241
CACTGAAAAGGAAAATGGGAATTTATAACCCAG



 


1
MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC
SEQ ID


61
SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC
NO: 38


121
RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW



181
GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH



241
QGWGKENCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



301
AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED



361
AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL



421
KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL



481
VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI



541
WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



601
LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG



661
DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR



721
LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP



781
IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE



841
VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD



901
TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL



961
NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH



1021
KEDAAVNCTD ISTRKTPQKA TTVSSRGENL VHQIQYREMN SCLNADDLNL MNSSGGHSEA



1081
H



 


1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H
SEQ ID NO:


1
ATGAGCAAACTCaGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT
39 and 40


 


21
F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



61
TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC



 


41
T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



121
ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT



 


61
S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



181
AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  R  T  A  I  K  A



241
AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC



 


101
T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  M  V  S  C



301
ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



361
CGTGGGAATGAGTCAGCTCTTTGGGACTOCAAACATGATGGATOGOGAAAGCATAGTAAC



 


141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



421
TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGG



 


161
L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  E  Q  G  Q  W



481
CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG



 


181
G  T  V  C  D  D  N  F  N  V  N  H  A  S  V  V  C  K  Q  L



541
GGAACAGTGTGTGATGATAACTTCAACGTCAATCATGCATCTGTGGTTTGTAAACAACTT



 


201
E  C  G  S  A  V  S  E  S  G  S  A  N  F  G  E  G  S  G  P



601
GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA



 


221
I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



661
ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT



 


241
Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



721
CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG



 


261
G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



781
GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA



 


281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



841
GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACACTCATGATGCT



 


301
A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



901
GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC



 


321
A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



961
GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGCACATGAACCT



 


341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGaT



 


361
A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  A  G  G  G  S



1081
GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC



 


381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
CGCTGTGCTGGGACAGTTGAGGTGGAGATtCAGAGACTGTTAGGGAAGGTGTGTGACAGA



 


401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC



 


421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261
AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT



 


441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT



 


461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381
ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG



 


481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC



 


501
S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG



 


521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC



 


541
W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA



 


561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC



 


581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG



 


601
L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT



 


621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861
TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA



 


641
G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA



 


661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981
GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA



 


681
I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041
ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA



 


701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC



 


721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161
TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG



 


741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG



 


761
G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC



 


781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGTATTTGGCAGTGCCATTCA



 


801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG



 


821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA



 


841
V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V



2521
GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG



 


861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L



2581
GGTGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA



 


881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC



 


901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701
ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG



 


921
T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761
ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA



 


941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG



 


961
N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA



 


981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941
GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC



 


1021
K  E  D  A  A  V  N  C  T  A  Q  K  I  S  T  H  K  T  P  Q



3061
AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA



 


1041
K  A  T  T  V  S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E



3121
AAAGCCACAACAGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAG



 


1061
M  N  S  C  L  N  A  D  D  L  D  L  M  N  S  S  G  G  H  S



3181
ATGAATTCTTGCCTGAATGCAGATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCT



 


1081
E  A  H  *  K  G  K  W  E  F  I  T  Q



3241
GAGGCACACTGAAAAGGAAAATGGGAATTTATAACCCAG



 


1
MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC
SEQ ID


61
SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC
NO: 40


121
RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW



181
GTVCDDNFNV NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH



241
QGWGKENCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



301
AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED



361
AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL



421
KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL



481
VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI



541
WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



601
LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG



661
DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR



721
LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP



781
IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE



841
VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD



901
TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL



961
NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH



1021
KEDAAVNCTA QKISTHKTPQ KATTVSSRGE NLVHQIQYRE MNSCLNADDL DLMNSSGGHS



1081
EAH



 


1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H 
SEQ ID NO:


1
ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT
41 and 42


 


21
F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



61
TTTGTCAACTTGAGTCCCTTCATTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC



 


41
T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



121
ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT



 


61
S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



181
AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



241
AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC



 


101
T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



301
ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



361
CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC



 


141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



421
TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGG



 


161
L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W



481
CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG



 


181
G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L



541
GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT



 


201
E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P



601
GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA



 


221
I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



661
ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT



 


241
Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



721
CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG



 


261
G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



781
GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA



 


281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



841
GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT



 


301
A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



901
GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC



 


321
A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



961
GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT



 


341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT



 


361
A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S



1081
GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC



 


381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGtGACAGA



 


401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC



 


421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261
AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT



 


441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT



 


461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381
ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG



 


481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC



 


501
S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG



 


521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC



 


541
W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA



 


561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC



 


581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG



 


601
L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT



 


621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861
TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA



 


641
G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA



 


661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981
GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA



 


681
I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041
ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA



 


701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC



 


721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  E  G  S  W



2161
TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG



 


741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG



 


761
G  C  G  E  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC



 


781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGTATTTGGCAGTGCCATTCA



 


801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG



 


821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA



 


841
V  F  Y  N  G  A  W  G  S  V  G  R  S  N  M  S  E  T  T  V



2521
GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG



 


861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L



2581
GGTGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA



 


881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC



 


901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701
ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG



 


921
T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761
ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA



 


941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG



 


961
N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA



 


981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941
GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC



 


1021
K  E  D  A  A  V  N  C  T  A  Q  K  I  S  T  H  K  T  P  Q



3061
AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA



 


1041
K  A  T  T  G  R  S  F  L  I  A  F  G  I  L  G  V  V  L  L



3121
AAAGCCACAACAGGTCGGTCATTCCTTATTGCATTCGGAATCCTTGGAGTTGTTCTCTTG



 


1061
A  I  F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V



3181
GCCATTTTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTT



 


1081
S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L



3241
TCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTG



 


1101
N  A  D  D  L  D  L  M  N  S  S  G  G  H  S  E  A  H



3301
AATGCAGATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCACAC



 


1
MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC
SEQ ID


61
SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC
NO: 42


121
RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW



181
GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH



241
QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



301
AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED



361
AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL



421
KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL



481
VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI



541
WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



601
LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG



661
DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR



721
LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP



781
IWLDEMKCNG KESRIWQCHS HGWGOQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE



841
VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD



901
TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL



961
NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH



1021
KEDAAVNCTA QKISTHKTPQ KATTGRSFLI AFGILGVVLL AIFVALFLTQ KRRQRQRLTV



1081
SSRGENLVHQ IQYREMNSCL NADDLDLMNS SGGHSEAH



 


1
M  S  K  L  R  M  V  L  L  E  D  S  G  S  A  D  V  R  R  H
SEQ ID NO:


1
ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT
43 and 44


 


21
F  V  N  L  S  P  F  T  I  A  V  V  L  L  L  R  A  C  F  V



61
TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC



 


41
T  S  S  L  G  G  T  T  K  E  L  R  L  V  D  G  E  N  K  C



121
ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT



 


61
S  G  R  V  E  V  K  I  Q  E  E  W  G  T  V  C  N  N  G  W



181
AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
S  M  E  A  V  S  V  I  C  N  Q  L  G  C  P  T  A  I  K  A



241
AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC



 


101
T  G  W  A  N  S  S  A  G  S  G  R  I  W  M  D  H  V  S  C



301
ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  S  N



361
CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC



 


141
C  T  H  Q  Q  D  A  G  V  T  C  S  D  G  S  D  L  E  M  R



421
TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGG



 


161
L  T  N  G  G  N  M  C  S  G  R  I  E  I  K  F  Q  G  Q  W



481
CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG



 


181
G  T  V  C  D  D  N  F  N  I  N  H  A  S  V  V  C  K  Q  L



541
GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT



 


201
E  C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P



601
GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA



 


221
I  W  F  D  D  L  I  C  N  G  N  E  S  A  L  W  N  C  K  H



661
ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT



 


241
Q  G  W  G  K  H  N  C  D  H  A  E  D  A  G  V  I  C  S  K



721
CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG



 


261
G  A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E



781
GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA



 


281
V  R  F  Q  G  E  W  G  T  I  C  D  D  G  W  D  S  H  D  A



841
GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT



 


301
A  V  A  C  K  Q  L  G  C  P  T  A  I  T  A  I  G  R  V  N



901
GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC



 


321
A  S  E  G  F  G  H  I  W  L  D  S  V  S  C  Q  G  H  E  P



961
GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT



 


341
A  V  W  Q  C  K  H  H  E  W  G  K  H  Y  C  N  H  N  E  D



1021
GCGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT



 


361
A  G  V  T  C  S  D  G  S  D  L  E  L  R  L  R  G  G  G  S



1081
GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC



 


381
R  C  A  G  T  V  E  V  E  I  Q  R  L  L  G  K  V  C  D  R



1141
CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTGTGTGACAGA



 


401
G  W  G  L  K  E  A  D  V  V  C  R  Q  L  G  C  G  S  A  L



1201
GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC



 


421
K  T  S  Y  Q  V  Y  S  K  I  Q  A  T  N  M  W  L  F  L  S



1261
AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT



 


441
S  C  N  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L



1321
AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT



 


461
T  C  D  H  Y  E  E  A  K  I  T  C  S  A  H  R  E  P  R  L



1381
ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG



 


481
V  G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G



1441
GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTGAAGTGAAGCATGGTGACACATGGGGC



 


501
S  V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q



1501
TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG



 


521
C  G  T  V  V  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I



1561
TGTGGCACAGTCGTCTCTATCCTGGGGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC



 


541
W  A  E  E  F  Q  C  E  G  H  E  S  H  L  S  L  C  P  V  A



1621
TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA



 


561
P  R  P  E  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y



1681
CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC



 


581
T  E  I  R  L  V  N  G  K  T  P  C  E  G  R  V  E  L  K  T



1741
ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG



 


601
L  N  A  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  L



1801
CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT



 


621
C  Q  Q  L  K  C  G  V  A  L  S  T  P  G  G  A  H  F  G  K



1861
TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA



 


641
G  N  G  Q  V  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G



1921
GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA



 


661
D  C  P  V  T  A  L  G  A  S  L  C  P  S  G  Q  V  A  S  V



1981
GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTGTA



 


681
I  C  S  G  N  Q  S  Q  T  L  S  S  C  N  S  S  S  L  G  P



2041
ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA



 


701
T  R  P  T  I  P  E  E  S  A  V  A  C  I  E  S  G  Q  L  R



2101
ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGOCCTGCATAGAGAGTGGTCAACTTCGC



 


721
L  V  N  G  G  G  R  C  A  G  R  V  E  I  Y  H  R  G  S  W



2161
TTGGTAAATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGG



 


741
G  T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L



2221
GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG



 


761
G  C  G  R  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P



2281
GGCTGTOGAGAGGCCATTAATGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGCGCCC



 


781
I  W  L  D  E  M  K  C  N  G  K  E  S  R  I  W  Q  C  H  S



2341
ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCCGTATTTGGCAGTGCCATTCA



 


801
H  G  W  G  Q  Q  N  C  R  H  K  E  D  A  G  V  I  C  S  E



2401
CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG



 


821
F  M  S  L  R  L  T  S  E  A  S  R  E  A  C  A  G  R  L  E



2461
TTCATGTCTCTGAGACTGACCAGTGAAGCCAGGAGAGAGGCCTGTGCAGGGCGTCTAGAA



 


841
V  F  Y  N  G  A  W  G  S  V  G  A  S  N  M  S  E  T  T  V



2521
GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG



 


861
G  V  V  C  R  Q  L  G  C  A  D  K  G  K  I  N  S  A  S  L



2581
GGTGTGGTGTGCAGGCAGCTGGGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTTTA



 


881
D  K  A  M  S  I  P  M  W  V  D  N  V  Q  C  P  K  G  P  D



2641
GACAAGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAAAAGGACCTGAC



 


901
T  L  W  Q  C  P  S  S  P  W  E  K  R  L  A  R  P  S  E  E



2701
ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGGCCAGGCCCTCGGAGGAG



 


921
T  W  I  T  C  D  N  K  M  R  L  Q  E  G  P  T  S  C  S  G



2761
ACCTGGATCACATGTGACAACAAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA



 


941
R  V  E  I  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L



2821
CGTGTGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTGTGTGATGACTCCTGGGACTTG



 


961
N  D  A  Q  V  V  C  Q  Q  L  G  C  G  P  A  L  K  A  F  K



2881
AACGATGCTCAGGTGGTGTGTCAACAACTTGGCTGTGGTCCAGCTTTGAAAGCATTCAAA



 


981
E  A  E  F  G  Q  G  T  G  P  I  W  L  N  E  V  K  C  K  G



2941
GAAGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGGG



 


1001
N  E  S  S  L  W  D  C  P  A  R  R  W  G  H  S  E  C  G  H



3001
AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC



 


1021
K  E  D  A  A  V  N  C  T  A  Q  K  I  S  T  H  K  T  P  Q



3061
AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA



 


1041
K  A  T  T  G  Q  S  F  L  I  A  F  G  I  L  G  V  V  L  L



3121
AAAGCCACAACAGGTCAGTCATTCCTTATTGCATTCGGAATCCTTGGAGTTGTTCTCTTG



 


1061
A  I  F  V  A  L  F  L  T  Q  K  R  R  Q  R  Q  R  L  T  V



3181
GCCATTTTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTT



 


1081
S  S  R  G  E  N  L  V  H  Q  I  Q  Y  R  E  M  N  S  C  L



3241
TCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTG



 


1101
N  A  D  D  L  D  L  M  N  S  S  E  N  S  N  E  S  A  D  F



3301
AATGCAGATGATCTGGACCTAATGAATTCCTCAGAAAATTCCAATGAGTCAGCTGATTTC



 


1121
N  A  A  E  L  I  S  V  S  K  F  L  P  I  S  G  M  E  K  E



3361
AATGCTGCTGAACTAATTTCTGTGTCTAAATTTCTTCCTATTTCTGGAATGGAAAAGGAG



 


1141
A  I  L  R  H  T  E  K  E  N  G  N  L



3421
GCCATTCTGAGGCACACTGAAAAGGAAAATGGGAATTTA



 


1
MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC
SEQ ID


61
SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC
NO: 44


121
RGNESALWDC KEDGWGKESN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW



181
GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH



241
QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA



301
AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED



361
AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL



421
KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL



481
VGGDIPCSGR VEVYHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI



541
WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT



601
LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG



661
DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR



721
LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP



781
IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE



841
VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD



901
TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL



961
NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH



1021
KEDAAVNCTA QKISTHKTPQ KATTGQSFLI AFGILGVVLL AIFVALFLTQ KRRQRQRLTV



1081
SSRGENLVHQ IQYREMNSCL NADDLDLMNS SENSNESADF NAAELISVSK FLPISGMEKE



1141
AILRHTEKEN GNL









Example 10
Cloning and Characterization of Canine CD163 from DH82 Cells
A forward primer 5′ simianCD163 (SEQ ID NO: 28) (5′-CACCGGAATGAGCAAACTCAGAATGG-3′ based on human CD163) and a reverse primer HuCD163-3′Kpn (SEQ ID NO: 29) (5′-GCTCCGGTACCTAGTCCAGGTCTTCA TCAAGGTATCTTA-3′) were used to amplify CD163 cDNA from DH82 cells. Total cellular RNA was prepared from DH82 cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer's instruction. Several clones containing large inserts were analyzed. Several clones with large inserts were analyzed, and these fell into either the v2 or v3 splicing patterns seen in other species. The v2 variant is missing an 81-nucleotide exon (E81) relative to the v3 variant, which results in a reading frame shift and alternative carboxy terminal amino acid sequences. The canine CD163v2 cDNA from DH82 cells encodes a peptide of 1115 amino acids. When compared to the sequences in Genbank database, it is 83.9% identical to human CD163 (Genbank Z22968), 85.1% identical to pig CD163 (Genbank AJ311716), and 74.3% identical to mouse CD163 (Genbank AF274883). The nucleotide and amino acid sequences of the two splice variants found in DH82 cells are provided below (SEQ ID NOS: 45-48).











SEQUENCE
ID NO

















1
M  S  K  L  R  M  V  P  H  G  N  S  G  S  A  D  F  R  R  C
SEQ ID NO:


1
ATGAGCAAACTCAGAATGGTCCCACATGGAAACTCTGGATCTGCTGACTTTAGAAGATGT
45 and 46


 


21
F  A  L  L  C  P  S  A  V  A  V  V  S  I  L  S  T  C  L  M



61
TTTGCCCTCTTGTGTCCCTCTGCTGTGGCTGTGGTCTCCATTCTCAGTACCTGTTTGATG



 


41
T  N  S  L  G  R  A  D  K  E  M  R  L  T  D  G  E  D  N  C



121
ACCAATTCTCTTGGGAGAGCAGATAAAGAGATGAGGCTAACGGATGGTGAAGACAATTGC



 


61
S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W



181
TCCGGGAGAGTGGAAGTGAAAGTCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
G  M  D  E  V  S  V  I  C  R  Q  L  G  C  P  T  A  I  K  A



241
GGCATGGATGAAGTCTCTGTGATTTGCAGGCAGCTGGGATGTCCCACTGCTATCAAAGCC



 


101
A  G  W  A  N  S  R  A  G  S  G  R  I  W  M  D  H  V  S  C



301
GCTGGATGGGCCAATTCCAGGGCAGGCTCTGGACGAATCTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C



361
CGAGGGAATGAATCTGCTCTCTGGGACTGCAAACATGATGGATGGGGAAAGCACAACTGC



 


141
S  H  Q  Q  D  A  G  V  T  C  S  D  G  S  S  L  E  M  R  L



421
AGTCATCAACAGGATGCTGGAGTAACCTGTTCAGATGGATCCAGTTTGGAGATGAGGTTG



 


161
M  N  G  G  N  Q  C  S  G  R  I  E  V  K  F  Q  G  Q  W  G



481
ATGAACGGCGGAAACCAGTGTTCTGGCAGAATAGAAGTCAAGTTCCAGGGACAGTGGGGA



 


181
T  V  C  D  D  N  F  N  I  D  H  A  S  V  V  C  K  Q  L  E



541
ACAGTGTGTGATGACAACTTCAACATAGATCATGCTTCTGTGGTTTGTAAACAGCTCGAA



 


201
C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P  I



601
TGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGTTCTGGGCCAATC



 


221
W  F  D  D  L  V  C  S  G  N  E  S  A  L  W  N  C  K  H  E



661
TGGTTTGATGATCTTGTGTGCAGTGGAAATGAGTCAGCTCTCTGGAACTGCAAGCATGAA



 


241
G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  D  G



721
GGATGGGGAAAGCATAACTGTGATCACGCTGAGGATGTTGGAGTGATTTGCTTGGATGGA



 


261
A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E  V



781
GCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAAGTA



 


281
K  F  Q  G  E  W  G  T  V  C  D  D  G  W  D  S  N  D  A  A



841
AAATTCCAAGGGGAATGGGGGACAGTGTGTGATGATGGCTGGGATAGTAATGATGCTGCT



 


301
V  V  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N  A



901
GTGGTATGTAAACAACTGGGATGCCCAACTGCTGTCACCGCCATTGGTCGAGTTAACGCC



 


321
S  E  G  S  G  H  I  W  L  D  N  L  S  C  Q  G  D  E  S  A



961
AGTGAGGGAAGTGGACACATTTGGCTTGACAATCTTTCCTGCCAAGGAGACGAATCTGCT



 


341
L  W  Q  C  R  H  H  E  W  G  K  H  Y  C  N  H  N  E  D  A



1021
CTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTATTGCAATCATAATGAAGATGCT



 


361
G  V  T  C  S  D  G  S  D  L  E  L  R  L  V  G  G  G  S  R



1081
GGTGTGACATGTTCTGATGGATCAGACCTGGAGCTGAGACTTGTCGGTGGAGGCAGCCGC



 


381
C  A  G  T  V  E  V  E  I  Q  K  L  L  G  K  V  C  D  R  G



1141
TGTGCTGGGACAGTGGAGGTTGAAATTCAGAAACTGCTAGGGAAAGTATGTGATAGAGGC



 


401
W  G  L  K  E  A  D  V  V  C  K  Q  L  G  C  G  S  A  L  K



1201
TGGGGACTGAAAGAAGCCGATGTGGTTTGCAAGCAGTTGGGATGTGGATCTGCTCTCAAA



 


421
T  S  Y  Q  R  Y  S  K  V  K  A  T  N  T  W  L  F  L  S  R



1261
ACGTCCTATCAGCGTTATTCCAAAGTTAAGGCAACAAACACATGGCTGTTTTTAAGCCGC



 


441
C  S  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L  S



1321
TGTAGTGGCAATGAAACTTCCCTTTGGGACTGCAAGAACTGGCAGTGGGGTGGACTGAGC



 


461
C  D  H  Y  E  E  A  K  V  T  C  S  A  H  R  E  P  R  L  V



1381
TGTGATCACTATGAAGAAGCTAAAGTTACCTGCTCAGCCCACAGGGAACCCAGACTAGTT



 


481
G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G  T



1441
GGAGGAGATATTCCCTGCTCTGGTCGTGTTGAAGTGAAACATGGTGACACATGGGGCACC



 


501
V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q  C



1501
GTCTGTGATTCCGACTTCTCTTTGGAAGCTGCCAGTGTGCTGTGCAGAGAGTTACAGTGT



 


521
G  T  V  I  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I  W



1561
GGCACAGTCATCTCCATCCTAGGGGGAGCTCACTTTGGAGAAGGAAATGGACAGATCTGG



 


541
A  E  E  F  Q  C  E  G  Q  E  S  H  L  S  L  C  S  V  A  S



1621
GCTGAAGAATTCCAGTGTGAGGGGCAGGAGTCCCATCTTTCACTCTGTTCAGTAGCCTCT



 


561
R  P  D  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y  T



1681
CGCCCAGATGGGACCTGTAGCCACAGCAGGGATGTTGGAGTCGTCTGCTCAAGATACACG



 


581
E  I  R  L  V  N  G  Q  S  P  C  E  G  R  V  E  L  K  I  L



1741
GAAATCCGCTTGGTGAATGGCCAGTCCCCGTGTGAAGGAAGAGTGGAGCTCAAGATACTT



 


601
G  N  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  F  C



1801
GGGAACTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCATGTTTTCTGT



 


621
Q  Q  L  K  C  G  V  A  L  S  I  P  G  G  A  H  F  G  K  G



1861
CAGCAGCTCAAATGTGGAGTTGCCCTTTCTATTCCGGGAGGAGCACATTTTGGGAAAGGA



 


641
S  G  Q  I  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G  D



1921
AGTGGTCAGATCTGGAGGCACATGTTTCACTGCACTGGGACTGAGCAGCACATGGGAGAT



 


661
C  P  V  T  A  L  G  A  T  L  C  S  A  G  Q  V  A  S  V  I



1981
TGCCCTGTAACTGCTCTGGGCGCGACGCTGTGTTCTGCTGGGCAAGTGGCCTCTGTAATC



 


681
C  S  G  N  Q  S  Q  T  L  S  P  C  N  S  T  S  L  D  P  T



2041
TGCTCAGGAAATCAGAGCCAGACGCTATCCCCATGCAATTCAACATCTCTGGACCCAACA



 


701
R  S  T  T  S  E  E  S  A  V  A  C  I  A  S  G  Q  L  R  L



2101
AGATCTACCACTTCGGAAGAAAGTGCTGTTGCTTGTATTGCGAGTGGGCAACTTCGCCTG



 


721
V  N  G  G  G  R  C  A  G  R  I  E  V  Y  H  E  G  S  W  G



2161
GTAAATGGAGGCGGTCGCTGTGCTGGGAGAATAGAGGTCTACCATGAGGGCTCCTGGGGC



 


741
T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L  G



2221
ACCATCTGTGATGACAGCTGGGACCTGAGTGATGCCCATGTGGTGTGCAGACAGCTGGGC



 


761
C  G  V  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P  I



2281
TGTGGAGTGGCCATTAATGCCACTGGCTCTGCTCATTTTGGGGAAGGAACAGGGCCCATC



 


781
W  L  D  E  V  N  C  N  G  K  E  S  H  I  W  Q  C  R  S  H



2341
TGGCTGGACGAGGTGAACTGTAATGGAAAGGAATCTCATATCTGGCAATGCCGCTCACAC



 


801
G  W  G  Q  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E  F



2401
GGCTGGGGGCAACACAACTGCAGACATAAGGAGGATGCAGGAGTTATCTGCTCAGAGTTC



 


821
M  S  L  R  L  I  D  E  T  S  R  D  I  C  A  G  R  L  E  V



2461
ATGTCTCTCAGACTGATTGATGAAACCAGCAGAGACATCTGTGCAGGGCGTCTTGAAGTT



 


841
F  Y  N  G  A  W  G  S  V  G  K  S  N  M  S  A  T  T  V  E



2521
TTTTACAATGGAGCTTGGGGCAGCGTTGGCAAGAGTAATATGTCTGCAACCACTGTGGAG



 


861
V  V  C  R  Q  L  G  C  A  D  K  G  S  I  N  P  A  S  S  D



2581
GTGGTATGCAGGCAACTGGGTTGTGCAGACAAGGGGAGCATCAACCCTGCATCTTCAGAC



 


881
K  P  M  S  R  H  M  W  V  D  N  V  Q  C  P  K  G  P  D  T



2641
AAGCCCATGTCCAGGCACATGTGGGTGGACAATGTCCAGTGTCCAAAAGGACCTGACACC



 


901
L  W  Q  C  P  S  S  P  W  K  Q  R  V  A  S  S  S  E  E  T



2701
TTATGGCAGTGCCCATCTTCTCCATGGAAACAGAGAGTGGCCAGTTCTTCAGAGGAGACC



 


921
W  I  T  C  A  N  K  I  R  L  Q  E  G  T  S  N  C  S  G  R



2761
TGGATCACATGTGCCAACAAGATAAGACTTCAAGAAGGAACCTCTAATTGTTCTGGACGT



 


941
V  E  L  W  H  G  G  S  W  G  T  V  C  D  S  S  H  D  L  E



2821
GTGGAGCTCTGGCACGGAGGTTCCTGGGGGACAGTGTGCGATGACTCCTGGGACCTTGAA



 


961
D  A  Q  V  V  C  R  Q  L  G  C  G  P  A  L  E  A  L  K  E



2881
GATGCACAAGTGGTGTGTCGACAGCTGGGCTGTGGCCCAGCATTAGAAGCACTAAAAGAG



 


981
A  A  F  G  Q  G  T  G  P  I  W  L  N  D  V  K  C  K  G  N



2941
GCAGCATTTGGTCAGGGGACTGGGCCTATATGGCTCAATGACGTGAAGTGCAAAGGGAAT



 


1001
E  S  S  L  W  D  C  P  A  R  P  W  G  H  S  D  C  G  H  K



3001
GAGTCTTCCTTGTGGGATTGTCCTGCTAGACCCTGGGGGCACAGTGACTGTGGCCACAAG



 


1021
E  D  A  A  V  R  C  S  E  I  A  M  A  Q  R  S  S  N  P  R



3061
GAAGATGCTGCTGTGAGGTGCTCAGAAATTGCAATGGCCCAAAGATCATCAAATCCTAGA



 


1041
G  H  S  S  L  V  A  L  G  I  F  G  V  I  L  L  A  F  L  I



3121
GGTCACTCATCCCTTGTTGCATTGGGGATCTTTGGTGTCATTCTTCTGGCCTTTCTCATC



 


1061
A  L  L  L  W  T  Q  R  R  R  Q  Q  Q  R  L  T  V  S  L  R



3181
GCTCTCCTCTTGTGGACTCAAAGGCGAAGACAGCAACAGCGGCTTACAGTTTCCTTGAGA



 


1081
G  E  N  S  V  H  Q  I  Q  Y  R  E  M  N  S  S  L  K  A  D



3241
GGAGAGAATTCTGTCCACCAAATTCAATACCGGGAAATGAATTCTTCCCTGAAAGCAGAT



 


1101
D  L  D  V  L  T  S  S  E  D  H  F  E  V  H



3301
GATCTGGACGTGCTGACTTCCTCAGAAGACCATTTTGACGTACAC



 


1
MSKLRMVPHG NSGSADFRRC FALLCPSAVA VVSILSTCLM TNSLGRADKE MRLTDGEDNC
SEQ ID


61
SGRVEVKVQE ENGTVCNNGW GMDEVSVICR QLGCPTAIKA AGWANSRAGS GRIWMDHVSC
NO: 46


121
RGNESALWDC KHDGWGKHNC SHQQDAGVTC SDGSSLEMRL MNGGNQCSGR IEVKFQGQWG



181
TVCDDNFNID HASVVCKQLE CGSAVSFSGS ANFGEGSGPI WFDDLVCSGN ESALWNCKHE



241
GWGKHNCDHA EDVGVICLDG ADLSLRLVDG VTECSGRLEV KFQGEWGTVC DDGWDSNDAA



301
VVCKQLGCPT AVTAIGRVNA SEGSGHIWLD NLSCQGDESA LWQCRHHEWG KHYCNHNEDA



361
GVTCSDGSDL ELRLVGGGSR CAGTVEVEIQ KLLGKVCDRG WGLKEADVVC KQLGCGSALK



421
TSYQRYSKVK ATNTWLFLSR CSGNETSLWD CKNWQWGGLS CDHYEEAKVT CSAHREPRLV



481
GGDIPCSGRV EVKHGDTWGT VCDSDFSLEA ASVLCRELQC GTVISILGGA HFGEGNGQIW



541
AEEFQCEGQE SHLSLCSVAS RPDGTCSHSR DVGVVCSRYT EIRLVNGQSP CEGRVELKIL



601
GNWGSLCNSH WDIEDAHVFC QQLKCGVALS IPGGAHFGKG SGQIWRHMFH CTGTEQHMGD



661
CPVTALGATL CSAGQVASVI CSGNQSQTLS PCNSTSLDPT RSTTSEESAV ACIASGQLRL



721
VNGGGRCAGR IEVYHEGSWG TICDDSWDLS DAHVVCRQLG CGVAINATGS AHFGEGTGPI



781
WLDEVNCNGK ESHIWQCRSH GWGQHNCRHK EDAGVICSEF MSLRLIDETS RDICAGRLEV



841
FYNGAWGSVG KSNMSATTVE VVCRQLGCAD KGSINPASSD KPMSRHMWVD NVQCPKGPDT



901
LWQCPSSPWK QRVASSSEET WITCANKIRL QEGTSNCSGR VELWHGGSWG TVCDDSWDLE



961
DAQVVCRQLG CGPALEALKE AAFGQGTGPI WLNDVKCKGN ESSLWDCPAR PWGHSDCGHK



1021
EDAAVRCSEI AMAQRSSNPR GHSSLVALGI FGVILLAFLI ALLLWTQRRR QQQRLTVSLR



1081
GENSVHQIQY REMNSSLKAD DLDVLTSSED HFEVH



 


1
M  S  K  L  R  M  V  P  H  G  N  S  G  S  A  D  F  R  R  C
SEQ ID


1
ATGAGCAAACTCAGAATGGTCCCACATGGAAACTCTGGATCTGCTGACTTTAGAAGATGT
NO: 47


 


21
F  A  L  L  C  P  S  A  V  A  V  V  S  I  L  S  T  C  L  M



61
TTTGCCCTCTTGTGTCCCTCTGCTGTGGCTGTGGTCTCCATTCTCAGTACCTGTTTGATG



 


41
T  N  S  L  G  R  A  D  K  E  M  R  L  T  D  G  E  D  N  C



121
ACCAATTCTCTTGGGAGAGCAGATAAAGAGATGAGGCTAACGGATGGTGAAGACAATTGC



 


61
S  G  R  V  E  V  K  V  Q  E  E  W  G  T  V  C  N  N  G  W



181
TCCGGGAGAGTGGAAGTGAAAGTCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG



 


81
G  M  D  E  V  S  V  I  C  R  Q  L  G  C  P  T  A  I  K  A



241
GGCATGGATGAAGTCTCTGTGATTTGCAGGCAGCTGGGATGTCCCACTGCTATCAAAGCC



 


101
A  G  W  A  N  S  R  A  G  S  G  R  I  W  M  D  H  V  S  C



301
GCTGGATGGGCCAATTCCAGGGCAGGCTCTGGACGAATCTGGATGGATCATGTTTCTTGT



 


121
R  G  N  E  S  A  L  W  D  C  K  H  D  G  W  G  K  H  N  C



361
CGAGGGAATGAATCTGCTCTCTGGGACTGCAAACATGATGGATGGGGAAAGCACAACTGC



 


141
S  H  Q  Q  D  A  G  V  T  C  S  D  G  S  S  L  E  M  R  L



421
AGTCATCAACAGGATGCTGGAGTAACCTGTTCAGATGGATCCAGTTTGGAGATGAGGTTG



 


161
M  N  G  G  N  Q  C  S  G  R  I  E  V  K  F  Q  G  Q  W  G



481
ATGAACGGCGGAAACCAGTGTTCTGGCAGAATAGAAGTCAAGTTCCAGGGACAGTGGGGA



 


181
T  V  C  D  D  N  F  N  I  D  H  A  S  V  V  C  K  Q  L  E



541
ACAGTGTGTGATGACAACTTCAACATAGATCATGCTTCTGTGGTTTGTAAACAGCTCGAA



 


201
C  G  S  A  V  S  F  S  G  S  A  N  F  G  E  G  S  G  P  I



601
TGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGTTCTGGGCCAATC



 


221
W  F  D  D  L  V  C  S  G  N  E  S  A  L  W  N  C  K  H  E



661
TGGTTTGATGATCTTGTGTGCAGTGGAAATGAGTCAGCTCTCTGGAACTGCAAGCATGAA



 


241
G  W  G  K  H  N  C  D  H  A  E  D  V  G  V  I  C  L  D  G



721
GGATGGGGAAAGCATAACTGTGATCACGCTGAGGATGTTGGAGTGATTTGCTTGGATGGA



 


261
A  D  L  S  L  R  L  V  D  G  V  T  E  C  S  G  R  L  E  V



781
GCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAAGTA



 


281
K  F  Q  G  E  W  G  T  V  C  D  D  G  W  D  S  N  D  A  A



841
AAATTCCAAGGGGAATGGGGGACAGTGTGTGATGATGGCTGGGATAGTAATGATGCTGCT



 


301
V  V  C  K  Q  L  G  C  P  T  A  V  T  A  I  G  R  V  N  A



901
GTGGTATGTAAACAACTGGGATGCCCAACTGCTGTCACCGCCATTGGTCGAGTTAACGCC



 


321
S  E  G  S  G  H  I  W  L  D  N  L  S  C  Q  G  D  E  S  A



961
AGTGAGGGAAGTGGACACATTTGGCTTGACAATCTTTCCTGCCAAGGAGACGAATCTGCT



 


341
L  W  Q  C  R  H  H  E  W  G  K  H  Y  C  N  H  N  E  D  A



1021
CTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTATTGCAATCATAATGAAGATGCT



 


361
G  V  T  C  S  D  G  S  D  L  E  L  R  L  V  G  G  G  S  R



1081
GGTGTGACATGTTCTGATGGATCAGACCTGGAGCTGAGACTTGTCGGTGGAGGCAGCCGC



 


381
C  A  G  T  V  E  V  E  I  Q  K  L  L  G  K  V  C  D  R  G



1141
TGTGCTGGGACAGTGGAGGTTGAAATTCAGAAACTGCTAGGGAAAGTATGTGATAGAGGC



 


401
W  G  L  K  E  A  D  V  V  C  K  Q  L  G  C  G  S  A  L  K



1201
TGGGGACTGAAAGAAGCCGATGTGGTTTGCAAGCAGTTGGGATGTGGATCTGCTCTCAAA



 


421
T  S  Y  Q  R  Y  S  K  V  K  A  T  N  T  W  L  F  L  S  R



1261
ACGTCCTATCAGCGTTATTCCAAAGTTAAGGCAACAAACACATGGCTGTTTTTAAGCCGC



 


441
C  S  G  N  E  T  S  L  W  D  C  K  N  W  Q  W  G  G  L  S



1321
TGTAGTGGCAATGAAACTTCCCTTTGGGACTGCAAGAACTGGCAGTGGGGTGGACTGAGC



 


461
C  D  H  Y  E  E  A  K  V  T  C  S  A  H  R  E  P  R  L  V



1381
TGTGATCACTATGAAGAAGCTAAAGTTACCTGCTCAGCCCACAGGGAACCCAGACTAGTT



 


481
G  G  D  I  P  C  S  G  R  V  E  V  K  H  G  D  T  W  G  T



1441
GGAGGAGATATTCCCTGCTCTGGTCGTGTTGAAGTGAAACATGGTGACACATGGGGCACC



 


501
V  C  D  S  D  F  S  L  E  A  A  S  V  L  C  R  E  L  Q  C



1501
GTCTGTGATTCCGACTTCTCTTTGGAAGCTGCCAGTGTGCTGTGCAGAGAGTTACAGTGT



 


521
G  T  V  I  S  I  L  G  G  A  H  F  G  E  G  N  G  Q  I  W



1561
GGCACAGTCATCTCCATCCTAGGGGGAGCTCACTTTGGAGAAGGAAATGGACAGATCTGG



 


541
A  E  E  F  Q  C  E  G  Q  E  S  H  L  S  L  C  S  V  A  S



1621
GCTGAAGAATTCCAGTGTGAGGGGCAGGAGTCCCATCTTTCACTCTGTTCAGTAGCCTCT



 


561
R  P  D  G  T  C  S  H  S  R  D  V  G  V  V  C  S  R  Y  T



1681
CGCCCAGATGGGACCTGTAGCCACAGCAGGGATGTTGGAGTCGTCTGCTCAAGATACACG



 


581
E  I  R  L  V  N  G  Q  S  P  C  E  G  R  V  E  L  K  I  L



1741
GAAATCCGCTTGGTGAATGGCCAGTCCCCGTGTGAAGGAAGAGTGGAGCTCAAGATACTT



 


601
G  N  W  G  S  L  C  N  S  H  W  D  I  E  D  A  H  V  F  C



1801
GGGAACTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCATGTTTTCTGT



 


621
Q  Q  L  K  C  G  V  A  L  S  I  P  G  G  A  H  F  G  K  G



1861
CAGCAGCTCAAATGTGGAGTTGCCCTTTCTATTCCGGGAGGAGCACATTTTGGGAAAGGA



 


641
S  G  Q  I  W  R  H  M  F  H  C  T  G  T  E  Q  H  M  G  D



1921
AGTGGTCAGATCTGGAGGCACATGTTTCACTGCACTGGGACTGAGCAGCACATGGGAGAT



 


661
C  P  V  T  A  L  G  A  T  L  C  S  A  G  Q  V  A  S  V  I



1981
TGCCCTGTAACTGCTCTGGGCGCGACGCTGTGTTCTGCTGGGCAAGTGGCCTCTGTAATC



 


681
C  S  G  N  Q  S  Q  T  L  S  P  C  N  S  T  S  L  D  P  T



2041
TGCTCAGGAAATCAGAGCCAGACGCTATCCCCATGCAATTCAACATCTCTGGACCCAACA



 


701
R  S  T  T  S  E  E  S  A  V  A  C  I  A  S  G  Q  L  R  L



2101
AGATCTACCACTTCGGAAGAAAGTGCTGTTGCTTGTATTGCGAGTOGGCAACTTCGCCTG



 


721
V  N  G  G  G  R  C  A  G  R  I  E  V  Y  H  E  G  S  W  G



2161
GTAAATGGAGGCGGTCGCTGTGCTGGGAGAATAGAGGTCTACCATGAGGGCTCCTGGGGC



 


741
T  I  C  D  D  S  W  D  L  S  D  A  H  V  V  C  R  Q  L  G



2221
ACCATCTGTGATGACAGCTGGGACCTGAGTGATGCCCATGTGGTGTGCAGACAGCTGGGC



 


761
C  G  V  A  I  N  A  T  G  S  A  H  F  G  E  G  T  G  P  I



2281
TGTGGAGTGGCCATTAATGCCACTGGCTCTGCTCATTTTGGGGAAGGAACAGGGCCCATC



 


781
W  L  D  E  V  N  C  N  G  K  E  S  H  I  W  Q  C  R  S  H



2341
TGGCTGGACGAGGTGAACTGTAATGGAAAGGAATCTCATATCTGGCAATGCCGCTCACAC



 


801
G  W  G  Q  H  N  C  R  H  K  E  D  A  G  V  I  C  S  E  F



2401
GGCTGGGGGCAACACAACTGCAGACATAAGGAGGATGCAGGAGTTATCTGCTCAGAGTTC



 


821
M  S  L  R  L  I  D  E  T  S  R  D  I  C  A  G  R  L  E  V



2461
ATGTCTCTCAGACTGATTGATGAAACCAGCAGAGACATCTGTGCAGGGCGTCTTGAAGTT



 


841
F  Y  N  G  A  W  G  S  V  G  K  S  N  M  S  A  T  T  V  E



2521
TTTTACAATGGAGCTTGGGGCAGCGTTGGCAAGAGTAATATGTCTGCAACCACTGTGGAG



 


861
V  V  C  R  Q  L  G  C  A  D  K  G  S  I  N  P  A  S  S  D



2581
GTGGTATGCAGGCAACTGGGTTGTGCAGACAAGGGGAGCATCAACCCTGCATCTTCAGAC



 


881
K  P  M  S  R  H  M  W  V  D  N  V  Q  C  P  K  G  P  D  T



2641
AAGCCCATGTCCAGGCACATGTGGGTGGACAATGTCCAGTGTCCAAAAGGACCTGACACC



 


901
L  W  Q  C  P  S  S  P  W  K  Q  R  V  A  S  S  S  E  E  T



2701
TTATGGCAGTGCCCATCTTCTCCATGGAAACAGAGAGTGGCCAGTTCTTCAGAGGAGACC



 


921
W  I  T  C  A  N  K  I  R  L  Q  E  G  T  S  N  C  S  G  R



2761
TGGATCACATGTGCCAACAAGATAAGACTTCAAGAAGGAACCTCTAATTGTTCTGGACGT



 


941
V  E  L  W  H  G  G  S  W  G  T  V  C  D  D  S  W  D  L  E



2821
GTGGAGCTCTGGCACGGAGGTTCCTGGGGGACAGTGTGCGATGACTCCTGGGACCTTGAA



 


961
D  A  Q  V  V  C  R  Q  L  G  C  G  P  A  L  E  A  L  K  E



2881
GATGCACAAGTGGTGTGTCGACAGCTGGGCTGTGGCCCAGCATTAGAAGCACTAAAAGAG



 


981
A  A  F  G  Q  G  T  G  P  I  W  L  N  D  V  K  C  K  G  N



2941
GCAGCATTTGGTCAGGGGACTGGGCCTATATGGCTCAATGACGTGAAGTGCAAAGGGAAT



 


1001
E  S  S  L  W  D  C  P  A  R  P  W  G  H  S  D  C  G  H  K



3001
GAGTCTTCCTTGTGGGATTGTCCTGCTAGACCCTGGGGGCACAGTGACTGTGGCCACAAG



 


1021
E  D  A  A  V  R  C  S  E  I  A  M  A  Q  R  S  S  N  P  R



3061
GAAGATGCTGCTGTGAGGTGCTCAGAAATTGCAATGGCCCAAAGATCATCAAATCCTAGA



 


1041
G  H  S  S  L  V  A  L  G  I  F  G  V  I  L  L  A  F  L  I



3121
GGTCACTCATCCCTTGTTGCATTGGGGATCTTTGGTGTCATTCTTCTGGCCTTTCTCATC



 


1061
A  L  L  L  W  T  Q  R  R  R  Q  Q  Q  R  L  T  V  S  L  R



3181
GCTCTCCTCTTGTGGACTCAAAGGCGAAGACAGCAACAGCGGCTTACAGTTTCCTTGAGA



 


1081
G  E  N  S  V  H  Q  I  Q  Y  R  E  M  N  S  S  L  K  A  D



3241
GGAGAGAATTCTGTCCACCAAATTCAATACCGGGAAATGAATTCTTCCCTGAAAGCAGAT



 


1101
D  L  D  V  L  T  S  S  E  Y  P  N  E  S  D  D  E  N  D  A



3301
GATCTGGACGTGCTGACTTCCTCAGAATATCCCAATGAGTCAGATGATTTTAATGATGCT



 


1121
G  L  I  S  V  S  K  S  L  P  I  S  G



3361
GGGCTAATTTCTGTGTCTAAATCTCTTCCTATTTCTGGA



 


1
MSKLRMVPHG NSGSADFRRC FALLCPSAVA VVSILSTCLM TNSLGRADKE MRLTDGEDNC
SEQ ID


61
SGRVEVKVQE EWGTVCNNGW GMDEVSVICR QLGCPTAIKA AGWANSRAGS GRIWMDHVSC
NO: 48


121
RGNESALWDC KHDGWGKHNC SHQQDAGVTC SDGSSLEMRL MNGGNQCSGR IEVKFQGQWG



181
TVCDDNFNID HASVVCKQLE CGSAVSFSGS ANFGEGSGPI WFDDLVCSGN ESALWNCKHE



241
GWGKHNCDHA EDVGVICLDG ADLSLRLVDG VTECSGRLEV KFQGEWGTVC DDGWDSNDAA



301
VVCKQLGCPT AVTAIGRVNA SEGSGHIWLD NLSCQGDESA LWQCRHHEWG KHYCNHNEDA



361
GVTCSDGSDL ELRLVGGGSR CAGTVEVEIQ KLLGKVCDRG WGLKEADVVC KQLGCGSALK



421
TSYQRYSKVK ATNTWLFLSR CSGNETSLWD CKNWQWGGLS CDHYEEAKVT CSAHREPRLV



481
GGDIPCSGRV EVKHGDTWGT VCDSDFSLEA ASVLCRELQC GTVISILGGA HFGEGNGQIW



541
AEEFQCEGQE SHLSLCSVAS RPDGTCSHSR DVGVVCSRYT EIRLVNGQSP CEGRVELKIL



601
GNWGSLCNSH WDIEDAHVFC QQLKCGVALS IPGGAHFGKG SGQIWRHMFH CTGTEQHMGD



661
CPVTALGATL CSAGQVASVI CSGNQSQTLS PCNSTSLDPT RSTTSEESAV ACIASGQLRL



721
VNGGGRCAGR IEVYHEGSWG TICDDSWDLS DAHVVCRQLG CGVAINATGS AHFGEGTGPI



781
WLDEVNCNGK ESHIWQCRSH GWGQHNCRHK EDAGVICSEF MSLRLIDETS RDICAGRLEV



841
FYNGAWGSVG KSNMSATTVE VVCRQLGCAD KGSINPASSD KPMSRHMWVD NVQCPKGPDT



901
LWQCPSSPWK QRVASSSEET WITCANKIRL QEGTSNCSGR VELWHGGSWG TVCDDSWDLE



961
DAQVVCRQLG CGPALEALKE AAFGQGTGPI WLNDVKCKGN ESSLWDCPAR PWGHSDCGHK



1021
EDAAVRCSEI AMAQRSSNPR GHSSLVALGI FGVILLAFLI ALLLWTQRRR QQQRLTVSLR



1081
GENSVHQIQY REMNSSLKAD DLDVLTSSEY PNESDDFNDA GLISVSKSLP ISG









Example 11
Various Cell Lines are Rendered Permissive to North American PRRSV Infection Following Transient Transfection with pCMV-susCD163v1
Porcine Kidney (PK032495), Norden Labs Swine Testicular (NLST-1), Norden Labs Dog Kidney (NLDK-1) were obtained from Pfizer Inc. and were grown at 37° C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Cell lines Baby Hamster Kidney (BHK21), Norden Labs Feline Kidney (NLFK-1), and Rabbit Lung (RL) were obtained from Pfizer Inc. and were grown at 37° C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Vero cells were obtained from Pfizer Inc. and were grown at 37° C. and 5% CO2 in growth media consisting of Minimum Essential Medium Alpha (MEM, Pfizer Inc. formulation) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and Gentamicin at 20 micrograms per mL. Cell culture wells (35 mm) containing approximately 1×106 cells were transfected with 2 micrograms per well of plasmid pCMV-susCD163v1, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. Cell line RL was transfected with 1.0 micrograms per well of plasmid pCMV-susCD163v1. A member of the PAM cell cDNA library without an insert, designated pPAMB (essentially and empty pSport plasmid vector), was used as a negative control plasmid. At 24 hours post transfection wells were aspirated and washed twice with DMEM/5% FBS followed by infection with North American PRRSV isolate P129. Virus was allowed to adsorb in 0.5 ml growth media for a minimum of two hours, after which additional media was added to a final volume of 2.0 ml and incubated overnight. The virus was then removed, wells washed twice with growth media, and fresh growth media added (2.0 ml per well). A time zero sample of culture fluid was immediately taken in order to determine the background level of infectious virus from the inoculum. At a minimum of 48 hours post infection cultures were screened for permissivity by removing culture fluids in order to assay viable virus, and permissive cells in the monolayer were detected by fluorescent antibody assay (FA). The FA was completed by fixing the monolayer with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for the PRRSV nucleocapsid protein. Viable virus was titrated by inoculating dilutions of culture fluids onto MARC-145 cells. Table 5 shows the results of virus infection by FA and the presence of progeny virus for each cell line tested.
Failure to detect progeny virus from some cell lines may be the result of low virus titer in the cell culture fluids, below the assay's limit of detection. Permissivity of Vero cells to PRRSV infection was augmented by the expression of susCD163v1. Compared to the time zero measurement of background virus, there was nearly a two-log increase in virus titers in Vero cells transfected with pCMV-susCD163v1, whereas there was less than a one-log in titer increase in cells transfected with negative control plasmid pPAMB. All cell lines except NLDK-1 were positive by FA for permissivity to North American PRRSV isolate P129 infection after transfection with pCMV-susCD163v1.







TABLE 5







Screening of various cell lines for permissivity


to NA PRRSV isolate P129 following transient


transfection with pCMV-susCD163v1 or pPAMB










Fluorescent Antibody assay
Progeny virus produced











Transfected
pCMV-

pCMV-



cell line
susCD163v1
pPAMB
susCD163v1
pPAMB





BHK21
+++
−
+++
−


PK032495
+
−
+
−


NKFK-1
+
−
+
−


NLST-1
+
−
−
−


NLDK-1
−
−
NT
NT


RL
+
−
−
−


Vero
++
+
++
+





+++ = Highly positive


++ = Moderately positive


+ = Slightly positive


− = Not detectible


NT = Not tested






Example 12
BHK21 Cells are Rendered Permissive to European PRRSV Infection Following Transient Transfection with pCMV-susCD163v1
Cell line Baby Hamster Kidney (BHK21) was obtained from Pfizer Inc. and grown at 37° C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Cell culture wells (35 mm) containing approximately 1×106 cells were transfected with 2 micrograms per well of plasmid pCMV-susCD163v1, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. At 24 hours post transfection wells were aspirated and washed twice with DMEM/5% FBS followed by infection with European PRRSV isolate 96V198. Virus was allowed to adsorb for a minimum of 2 hours. The virus was then removed, wells washed twice with growth media, and fresh growth media added (2.0 ml per well). A time zero sample of culture fluid was immediately taken in order to determine the background level of infectious virus from the inoculum. At a minimum of 48 hours post infection cultures were screened for permissivity by removing culture fluids in order to assay viable virus, and permissive cells in the monolayer were detected by fluorescent antibody assay (FA). The FA was completed by fixing the monolayer with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for the PRRSV nucleocapsid protein. Viable virus was titrated by inoculating dilutions of culture fluids onto MARC-145 cells. As a result of the transient transfection of BKH21 with pCMV-susCD163v1, cells were rendered permissive to European PRRSV isolate 96V198 infection and yielded progeny virus.
Example 13
CD163 Genes from Multiple Animal Species Render BHK21 Cells Permissive to PRRS Virus Infection
BHK21 cells grown in DMEM (Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics, were used in transient transfection experiments. Before transfection cells were washed once with OptiMEM (Invitrogen) without serum or other additives. Lipofectamine 2000 (Invitrogen) was used in all transfection experiments according to the protocol provided by the manufacturer. The transfection mixture consisted of 10 microliters of Lipofectamine 2000 and 2-3 micrograms of DNA per 35 mm well. After overnight incubation, transfection medium was removed and cells were infected with PRRSV isolate P129. Infection was allowed to progress for 24-48 hours, when cells were fixed with 80% acetone and stained with monoclonal antibody SDOW17 conjugated with FITC (Rural Technology Inc., Brookings, S. Dak.). Staining of the nucleocapsid protein was visualized under a fluorescence microscope. Table 6. Transient transfection of BHK21 cells with various CD163 genes renders them permissive to PRRS virus infection











TABLE 6









PRRSV infection



Plasmid backbone
CD163 gene
(FA)









pCMV-Script
Swine CD163v1
+++



pRSV-Script
Swine CD163v1
+++



pcDNA3.1D
Swine CD163v2
++



pcDNA3.1D
Human CD163v2
++



pcDNA3.1D
Mouse CD163v3
+



pcDNA3.1D
African green monkey
+++




(MARC-145 cell)




CD163v2



pcDNA3.1D
Vero cells CD163v7
+++



pcDNA3.1D
DH82 cell CD163v2
+++







+++ = Highly positive



++ = Moderately positive



+ = Slightly positive






Example 14
Generation of PRRSV-Permissive BHK21 Stable Cell Lines Using pCMV-susCD163v1
BHK-21 cells were grown in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics. For transfection, cells were seeded at approximately 90% confluency in 6 well plates and incubated over night at 37° C. in 5% CO2. Cells were transfected with pCMV-susCD163v1 DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. One day after transfection the cells were trypsinized and re-seeded in 96 well plates in a dilution series. To select for stable transfectants, the media was supplemented with 1 mg/ml Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) from this point forward. Medium was changed every 3-5 days. Plates were cultured until those wells with colonies derived from single cells reached confluency, at which point the plates were trypsinized and seeded into duplicate 96 well plates. One of the duplicate 96 well plates was infected with PRRSV isolate P129 and clones permissive to infection were identified by staining with FITC conjugated monoclonal antibody SDOW17. Positive clones were then expanded from the second duplicate plate. To ensure homogeneity the positive cultures were single-cell cloned by limiting dilution. At each cloning the subclones that displayed robust growth and high PRRSV permissivity were chosen for expansion. Three clones designated BHK/CMV/v1 #3, BHK/CMV/v1 #5, and BHK/CMV/v1 #12 (FIG. 6) were selected. These cell lines have maintained the permissive phenotype through 20 passages.
Example 15
Generation of PRRSV-Permissive BHK21 Stable Cell Lines Using pRSV-susCD163v1
BHK-21 cells were cultured as described in Example 14. BHK-21 cells were transfected with pRSVsusCD163v1 using Lipofectamine 2000 as described in Example 14. Cloning of transfected cells and screening for permissive clones was performed essentially as described in Example 14. From the original cloning 3 single cell clones were identified as permissive and were subsequently recloned two more times to ensure homogeneity and to attempt to isolate subclones of higher permissivity (see FIG. 7). The resulting cell lines were named BHK/RSV/v1, #2, #3, and #4. All of these clones have maintained the permissive phenotype through the highest passage tested (passage 11 for clone #2, passage 7 for clone #3, and passage 5 for clone #4).
Example 16
Generation of PRRSV-Permissive Feline Kidney Stable Cell Lines Using pCMV-susCD163v1
Parental Norden Labs Feline Kidney (NLFK) cells were grown at 37 degrees C. and 5% CO2 in Dulbecco's Modified Eagle Medium (Invitrogen catalog number 11965-092) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics. Several 35 mm wells containing approximately 2×106 cells each were transfected with 4 micrograms per well of pCMV-susCD163v1, in OptiMEM, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were removed from the substrate using Accutase (Innovative Cell Technologies, catalog number AT104) diluted in medium, and seeded into three 96-well plates at three densities (approximately 2×102, 2×103, and 2×104 cells per well). The cells were allowed to settle overnight at 37 degrees C. before beginning selection of stable transformants. The next morning medium was replaced with 100 microliters/well fresh medium containing 500 micrograms/ml Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) to select for cells expressing the neomycin resistance gene. Medium was changed every 2 or 3 days to maintain Geneticin potency. After 19 days of selection, the 96-well plate with the lowest initial cell density (approximately 200 cells/well) yielded 70 empty wells and 26 wells with one or more colonies of G418-resistant cells (calculated number of resistant cells/well is 0.3, using the Poisson distribution). These 26 wells were split into duplicate wells and allowed to settle overnight. One set of wells was infected with PRRSV isolate P129, incubated for 24 hours, then fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid. Of the 26 clones, 8 contained some cells that were infected by PRRSV. One of these, designated “NLFK-CMV-susCD163v1-G4”, was clearly more permissive than the others with nearly 100% of the cells staining positive for viral antigen.
By cell passage number 5, there was some evidence of phenotypic heterogeneity in the NLFK-CMV-susCD163v1-G4 cell line. Therefore, the cells were single-cell cloned by limiting dilution in G418-containing medium, starting with frozen stocks of NLFK-CMV-susCD163v1-G4 passage 4. Twelve such clones (“A”-“L”) were expanded for study. Of these, clones NLFK-CMV-susCD163v1-G4F and NLFK-CMV-susCD163v1-G4L were notable for their ability to form discrete plaques (localized areas of CPE) when infected with PRRSV isolate P129 (see FIG. 8).
Example 17
Generation of PRRSV-Permissive Feline Kidney Stable Cell Lines Using pRSV-susCD163v1
Norden Labs Feline Kidney (NLFK) cells were grown at 37° C. and 5% CO2 in Minimal Essential Medium Alpha Medium (Invitrogen catalog number 12571-071) supplemented with 10% fetal bovine serum and antibiotics. NLFK cells were seeded in 6 well plates at approximately 90% confluency and allowed to attach overnight. The cells were then transfected with plasmid pRSV-susCD163v1 using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. After 24 hours the cells were cloned as described in Example 14. Screening for PRRSV permissive cell clones was performed as described in Example 14. Four clones were selected from the screening and were single cell cloned by limiting dilution two more times. Four clones named FK/RSV/v1 #1, FK/RSV/v1 #2, FK/RSV/v1 #3, and FK/RSV/v1 #4 were selected. These cell lines have maintained the PRRSV permissive phenotype through at least 8 passages (see FIG. 9).
Example 18
Generation of PRRSV-Permissive Porcine Kidney Stable Cell Lines Using pCMV-susCD163v1
Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1×106 cells each were transfected with 2 micrograms per well of plasmid pCMV-susCD163v1, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and allowed to incubate until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum of 48 hours. Eleven clones were found to be permissive for PRRSV. One of these, designated “PK-CMV-susCD163v1-A10”, clearly retained the permissive phenotype after numerous passages (see FIG. 10).
Example 19
Generation of PRRSV-Permissive BHK21 Stable Cell Lines Using pCMVScript-susCD163v2
Parental Baby Hamster Kidney (BHK21) cells were obtained from Pfizer Inc. and were grown at 37 degrees C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1×106 cells each were transfected with 2 micrograms per well of pCMVScript-susCD163v2, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSV isolate P129 and incubating for a minimum of 48 hours. Three clones were found to be PRRSV-permissive, and one of these, designated “BHK-CMVScript-susCD163v2-A9”, was chosen for further study (see FIG. 11).
Example 20
Generation of PRRSV-Permissive BHK-21 Stable Cell Lines Using pRSV-susCD163v2
BHK-21 cells were cultured as described in Example 14. BHK-21 cells were transfected with the ligated pRSV-susCD163v2 DNA construct described in Example 5 using Lipofectamine 2000 (Invitrogen) following manufacture's instructions. Subsequent cloning and selection of PRRSV permissive cell lines was performed as described in Example 14. Of 336 single cell clones screened, 129 were positive. Several of these cell clones have been passed up to 7 times and they have maintained the PRRSV permissive phenotype (see FIG. 12). These cell lines have been named BHK/RSV/v2, followed by a numerical clone number.
Example 21
Generation of PRRSV-Permissive Porcine Kidney Stable Cell Lines Using pCMVScript-susCD163v2
Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1×106 cells each were transfected with 2 micrograms per well of pCMVScript-susCD163v2 in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSV isolate P129 and incubating for a minimum 48 hours. One clone designated “PK-CMVScript-susCD163v2-D1” showed the PRRSV-permissive phenotype.
Example 22
Generation of PRRSV-Permissive BHK21 Stable Cell Lines Using pcDNA3.1D-humCD163v2
Parental Baby Hamster Kidney (BHK21) cells were obtained from Pfizer Inc. and were grown at 37 degrees C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1×106 cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSV isolate P129, incubated for a minimum 48 hours. Seven candidate clones were found to be PRRSV-permissive. There was some evidence of phenotypic heterogeneity in each of the seven candidate clones, likely because they were not clonal. Therefore, the candidate clones were single-cell cloned by limiting dilution in G418 containing medium. One single cell clone with clear PRRS-permissivity was obtained and designated BHK-cDNA3.1D-humCD163v2-H9.
Example 23
Generation of PRRSV-Permissive Feline Kidney Stable Cell Lines Using pcDNA3.1D-humCD163v2
Parental Norden Labs Feline Kidney (NLFK) cells were grown at 37 degrees C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1×106 cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2 in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104), diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 500 micrograms per ml, and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum 48 hours. Five clones were found to be permissive. One of these, designated “FK-cDNA3.1D-humCD163v2-A6”, clearly displayed the permissive phenotype (see FIG. 13).
NLFK parent cells and one subclone of FK-cDNA3.1D-humCD163v2-A6 were examined for the CD163 expression. Cells were fixed in 80% acetone and reacted with Goat anti-human CD163 (R&D System at 1:200) for one hour following by washing with PBS. For visualization, donkey anti-Goat IgG conjugated with FITC (Biodesign Inc at 1:100) were used. No specific fluorescence was detected in the NLFK parent cells as shown in FIG. 21A. The majority of the FK.A6.A2 subclone showed good fluorescent staining indicating the presence of CD163 (FIG. 21B).
Example 24
Generation of PRRSV-Permissive Porcine Kidney Stable Cell Lines Using pcDNA3.1D-humCD163v2
Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C. and 5% CO2 in growth media consisting of Dulbecco's Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1×106 cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104), diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml, and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum 48 hours. Two clones were found to be permissive. One of these, designated “PK-cDNA3.1D-humCD163v2-B11” clearly showed the PRRSV-permissive phenotype.
Example 25
Generation of PRRSV-Permissive Feline Kidney Stable Cell Line Using Ligated pRSV-Script MARC CD163v2
A non-cloning based procedure to generate microgram quantities of linear DNA suitable for use in generating stable cell lines expressing CD163 from an RSV promoter was developed (FIG. 4). A similar process was adapted to place simian CD163v2 from MARC-145 cells behind the RSV promoter. The procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and RSV promoter cassette derived from pRSV-script, and the other containing the MARC CD163v2 coding sequence from pCDNA3.1D MARC CD163v2. Vector plasmid pRSV-Script was linearized with Hind III and Kpn I. Plasmid was first digested with Kpn I and was bunted with the Klenow fragment of E. coli DNA polymerase. This plasmid was then digested with Hind III immediately downstream of the RSV promoter. The pCDNA3.1D MARC CD163v2 clone was digested in the vector sequence downstream of the CD163 insert with EcoRV, and Hind III upstream of CD163. The CD163 coding sequence was liberated from the vector. For each plasmid digestion the appropriate fragments were purified from agarose gels. A large-scale ligation reaction was performed as follows. Approximately 20 μg of each DNA fragment was incubated in a volume of 600 μL with 15 units of T4 DNA ligase. The reaction was incubated at room temperature for 1 hour. Following ligation, a linear piece of DNA containing all of the appropriate elements was purified by agarose gel electrophoresis. Restriction enzyme digestion analysis was performed to confirm the authenticity of each ligated fragment. Ligation of the two DNA fragments via the cohesive Hind III termini resulted in the placement of the 5′ sequences of the MARC CD163 gene downstream of the RSV promoter, allowing for directed expression of CD163 in mammalian cells. Once isolated, the purified DNA was used to transfect selected mammalian cell lines.
Norden Labs Feline Kidney (NLFK) cells were grown at 37° C. and 5% CO2 in DMEM supplemented with 5% fetal bovine serum and antibiotics. NLFK cells were seeded in 6 well plates at approximately 90% confluency and allowed to attach overnight. The cells were then transfected with ligated plasmid pRSV-MARC CD163v2 using Lipofectamine 2000 following the manufacturer's instructions. After 24 hours the cells were cloned as described in Example 12. Screening for PRRSV permissive cell clones was performed as described in Example 12. One clone was positive for PRRSV infection and designated NLFK-MARC CD163 D4. This D4 clone has maintained the PRRSV permissive phenotype through 9 passages.
Example 26
Growth Kinetics of PRRSV Isolate NVSL 94-3 in Recombinant BHK-21 and NLFK Cells Stably Expressing susCD163v1 from the CMV Promoter
The amounts of progeny virus produced by PRRSV-infected BHK-21 or NLFK cells stably engineered to express susCD163v1 were quantitated. Four cell lines expressing susCD163v1, BHK/CMV/susv1 #3, BHK/CMV/susv1 #5, BHK/CMV/susv1 #12, and FK/CMV/susv1 G4 were seeded at sub confluency in 6 well plates and, after overnight incubation, were infected with the NVSL 94-3 isolate of PRRSV. MARC-145 cells were included in the experiment for comparison. The cells were infected with virus at an m.o.i. of approximately 0.1. Virus was adsorbed for 60-90 minutes and was removed. Cells were washed three times with PBS to remove residual virus. One-milliliter aliquots were harvested from the cultures at 12-hour intervals starting immediately after infection and continued through 96 hrs. Fresh culture media was added to the cells at various time points to maintain a culture volume sufficient to prevent the cell monolayer from drying out. Culture supernatants were stored at −80° until all samples were collected. The amount of PRRSV present in the culture supernatants was determined by plaque assay on MARC-145 cells. FIG. 14 shows that all CD163 expressing recombinant cell lines tested were able to produce progeny PRRSV.
Example 27
Blocking PRRSV Infection with Anti-CD163 Antibody
Transiently Transfected Cells
BHK-21 cells, seeded in 24 well plates, were transiently transfected with the plasmid pCDNA3.1D-MARC-CD163v2 described in example 8, using Lipofectamine 2000 as described in example 14. After overnight incubation to allow expression of CD163, a titration of goat polyclonal antibody specific for human CD163 (R&D Systems, cat #AF1607) in PBS was added to the cells in a volume of 100 μl. As a control, equivalent amounts of normal goat IgG (R&D Systems, cat #AB-108-C) were used. Following a one-hour incubation at 37° C., the monolayers were infected with approximately 1×107 pfu of a recombinant P129 strain of PRRSV that expresses GFP. The cell monolayers, with anti-CD163 antibody and PRRSV, were incubated at 37° C. for one hour at which time the virus inoculum/antibody mixture was aspirated, the cell monolayer washed once with PBS, and 1 ml of growth medium added to the wells. The cells were incubated for 24 hours at 37° C. to allow PRRSV directed GFP expression. For analysis, the cells were trypsinized, resuspended in 500 μl of PBS and analyzed by flow cytometry to innumerate the PRRSV infected cells via GFP expression. For flow cytometry, uninfected BI-IK-21 cells were used to set the baseline for fluorescence detection, and approximately 100,000 cells were analyzed from each subsequent sample. The results of this analysis, shown in FIG. 15, show that the CD163 specific antibody was able to significantly reduce the number of infected cells when compared to cells incubated with normal goat IgG.
Example 28
Blocking PRRSV Infection by Anti CD163 Antibody
Stably Transfected Cells
The NLFK cells that stably express human CD163 (FK-cDNA3.1D-humCD163v2-A6), described in Example 23, were seeded into 24 wells plates. After allowing the cells to attach overnight, a titration of goat polyclonal antibody specific for human CD163 (R&D Systems, cat #AF1607) in PBS was added to the cells in a volume of 100 μl. As a control, equivalent amounts of normal goat IgG (R&D Systems, cat #AB-108-C) were used. Following a one-hour incubation at 37° C., the monolayers were infected with approximately 1×107 pfu of a recombinant P129 strain of PRRSV that expresses GFP. The cell monolayers, with anti-CD163 antibody and PRRSV, were incubated at 37° C. for one hour at which time the virus inoculum/antibody mixture was aspirated, the cell monolayer washed once with PBS, and 1 ml of growth medium added to the wells. The cells were incubated for 24 hours at 37° C. to allow PRRSV directed GFP expression. For analysis, the cells were trypsinized, resuspended in 500 μl of PBS, and analyzed by flow cytometry to innumerate the PRRSV infected cells via GFP expression. Approximately 100,000 cells were analyzed from each sample. The results of this analysis, shown in FIG. 16, show that the CD163 specific antibody was able to significantly reduce the number of infected cells when compared to cells incubated with normal goat IgG.
Example 29
Generation of PRRSV-Permissive Porcine Kidney Stable Cell Lines Using pRSV-susCD163v2
Porcine kidney cells (PK032495) were cultured as described in Example 21. For transfection, cells were seeded in a 24 well plate at 80% confluency and allowed to recover overnight. Transfection of ligated pRSV-susCD163v2 DNA described in Example 5 was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Subsequent cloning and selection of PRRSV permissive cells was performed essentially as described in Example 14. The initial cloning by limiting dilution failed to yield single cell derived clones, so 5 wells with PRRSV permissive cells were recloned by limiting dilution to yield clonal cell lines. 10 clones were selected for further study and one of these clones, PK-RSVScript-susCD163v2 #9 showed the ability to support foci growth of PRRSV early after infection (see FIG. 18).
Example 30
Generation of PRRSV-Permissive Feline Kidney Stable Cell Lines Using pRSV-susCD163v2
NLFK feline kidney cells were cultured as described in Example 17. For transfection, cells were seeded at approximately 80% of maximal density in 24 well plates. After overnight incubation the monolayers were transfected with ligation derived RSV/susCD163v2 (see example 5) using Lipofectamine following the manufacturer's instructions. Cloning of the transfected cells and selection of PRRSV permissive cell clones was performed essentially as described in Example 14. Of the 67 cell clones tested for PRRSV permissivity, 20 were found to be positive. An example of the staining observed is shown in FIG. 19.
Example 31
Passage of PRRSV Isolate P201 in PK-RSVScript-susCD163v2 Cells
Amplification of a PRRSV clinical isolate was performed as follows. Peripheral alveolar macrophage (PAM) cells were seeded at 5.4E6 cells per 10 cm2 in a 6 well dish using OptiMEM media supplemented with 2% FBS. After 6 hours the media was aspirated and a 2 ml aliquot of serum harvested from a PRRSV infected pig was added to the cells. Following a 90 minute adsorption, the serum inoculum was removed and replaced with OptiMEM. At approximately 40 post infection the supernatant was harvested and clarified with a 10 minute centrifugation. The supernatant was directly used to infect PK-RSVScript-susCD163v2 clone #9 cells using a 6 hour adsorption. After removal of the inoculum the cells were refed with D-MEM. The P201 virus was serially passaged on the PK-RSVScript-susCD163v2 #9 cell line using alternating infected cell and cell free supernatant passes. We observed that for efficient spread of the virus, the cells should be seeded at 50-70% confluency the day before infection, using flasks of cells that were kept at sub-confluency. To follow the progression of infection, each passage was replicated in multiple wells of identically infected cells and at each day one of the wells was acetone fixed and stained with the FITC labeled monoclonal antibody SDOW17. If the percentage of infected cells was not greater than 50% and significant progression of foci development over the prior clays observations were not seen, the cells in an equivalent well were trypsinized and passed to multiple fresh wells. These infected cell passages were typically at a 1:4 split and sometimes included the addition of an equivalent number of cells from an uninfected culture. Alternatively, if the SDOW17 staining revealed that the infected cell foci had spread sufficiently to account for greater than 50% of the total cells, cell free supernatant was harvested and used to infect multiple wells of freshly seeded cells (FIG. 20). After 11 passages the intervening cell passages were not necessary as the virus was able to grow to sufficient titer to allow consecutive cell free supernatant passaging of the virus.
Example 32
Screening Various CD163 Cell Lines for Permissivity to Various European and North American PRRSV Isolates
Various CD163 transgenic cell lines were assessed for permissivity to low passage European and North American PRRSV isolates (see Table 7). Transgenic CD163 cell lines as described in earlier examples included NLFK-MARC CD163 D4, PK-RSVScript-susCD163v2clone #9 and PK-CMV-susCD163v1-A10. Each CD163 cell line along with cell lines MARC-145, parental feline kidney, parental porcine kidney cell lines (serving as controls) were planted onto 96-well tissue culture plates. Growth media was removed from monolayers and inoculated with 0.1 mL per well of each PRRSV isolate. At day 3 post infection, the plates were fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc.) which is specific for the nucleocapsid. Results of the fluorescent antibody (FA) assay are in Table 7. Table 7










FA results of screening of various CD163 cell lines for permissivity


to European and North American PRRSV isolates









PRRSV Isolatea













CD163 Cell Line
EU98V226
P129
P201
1151
94-3
IND5





NLFK-MARC
++
+
+++
+++
++
++++


CD163 D4


PK-RSVScript-
+
+
++
+
+
++


susCD163v2clone #9


PK-CMV-
+
+
++
++
++
++


susCD163v1-A10


MARC-145
++
+
+++
+
++++
+++


Porcine Kidney
−
−
−
−
−
−


(parental)


Feline Kidney
−
−
−
−
−
−


(parental)





aAll PRRSV isolates are North American except EU98V226 is a European isolate.






Example 33
Phorbol 12-myristate 13-acetate (PMA) Induction of CD163 Renders Human U937 Cells Permissive to PRRSV Infection
Human U937 cells obtained from ATCC (CRL-1593.2) were propagated in RPMI medium containing serum and additives according to ATCC specifications. These cells are known to express CD163 when activated by PMA treatment (Gronlund et al., 2000). U937 cells were seeded in duplicate in wells of a 6-well plate. One set of wells was treated with 100 ng/ml of PMA and the other set was left untreated. Three days after PMA stimulation, one well from each set was infected with the P129 isolate of PRRSV. The other well from each set was fixed and stained for expression of CD163 in an indirect immunofluorescent antibody assay using goat-anti human CD163 (R&D System) and donkey anti-goat IgG conjugated with FITC (BioDesign International).
Untreated U937 cells continued propagation to high density 3 days after initial planting. PMA-treated U937 cells stopped propagating, became enlarged, and attached to the surface of the culture wells. A small fraction of untreated U937 were positive for CD163 staining, whereas almost all PMA-treated U937 were positive for CD163 staining. In untreated U937 no PRRSV infected cells were observed. However, hundreds of PMA treated U937 cells became infected by PRRSV. This demonstrates that non-permissive cells can be rendered permissive for PRRSV infection following chemical induction of CD163 expression.
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations that are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations that have not been described herein as critical are intended as aspects of the invention.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention.
The entire disclosure of all publications cited herein are hereby incorporated by reference to the extent they are not inconsistent to the disclosure herein.

## Claims

1. A method of facilitating production of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) from a culture of vertebrate cells, comprising the steps of:
(a) providing a recombinant vertebrate cell transfected with an exogenous polynucleotide that encodes a polypeptide having a transmembrane domain and at least 90% sequence identity to SEQ ID NO:24 or SEQ ID NO:27, so that expression of CD163 polypeptide in said cell is increased;
(b) contacting a culture of said cell with PRRSV virus under conditions which permit infection of the cells and growth of the virus; and
(c) recovering virus from said culture.

2. The method of claim 1, wherein the cell was previously PRRSV permissive and is rendered more PRRSV permissive.

3. The method of claim 2, wherein the cell did not previously express a CD163 polypeptide and is induced to express CD163.

4. The method of claim 3, wherein the cell is selected from the group consisting of baby hamster kidney cells (BHK21), porcine kidney cells, feline kidney cells, avian cells, and swine testicular cells.

5. The method of Claim 1, wherein the PRRSV is of the European genotype.

6. The method of claim 1, wherein the PRRSV is of the North American genotype.

7. The method of claim 1, wherein the polynucleotide encodes a polypeptide having a transmembrane domain and at least 95% sequence identity to SEQ ID NO:24 or SEQ ID NO:27.

8. The method of claim 1, wherein the polynucleotide encodes a apolypeptide having a transmembrane domain and at least 99% sequence identity to SEQ ID NO:24 or SEQ ID NO:27.

9. The method of claim 1, further comprising the step of producing a vaccine from the recovered virus.