# Patent

## Title
Gene expression in plants

## Abstract

This application is a Divisional application under 37 C.F.R.I.62 of prior application Ser. No. 08/138,609, filed Oct. 15, 1993, which is a continuation of Ser. No. 07/722,971 filed Jun. 28, 1991, both now abandoned.

## Background
The present invention is in the veterinary field. More specifically, the invention relates to the prophylaxis and therapy of pleuropneumonia in pigs.

Pleuropneumonia is a major respiratory disease in pigs and causes severe economic losses in pig farming in many countries including the United States and Canada. The disease is caused by the bacterium Actinobacillus pleuropneumoniae (previously also referred to as Haemophilus pleuropneumoniae) and is considered to be one of the most important disorders of the bronchial tubes in pigs. Frequently, the disease is fatal. Actinobacillus pleuropneumoniae is known to exist in twelve infective serotypes.

Since pleuropneumonia can be induced by inoculating pigs with sterile culture supernatants of A. pleuropneumoniae, extracellular toxic proteins are assumed to be involved in the development of the pneumonic lesions. There is growing evidence that qualitative or quantitative differences in toxic activities exist between the twelve serotypes of A. pleuropneumoniae. Hemolytic and cytotoxic activities have been reviewed by T. A. Bertram, Can. J. Vet. Res. 54: 553-556 (1990). Two different hemolytic activities were reported by Frey and Nicolet, J. Clin. Microbiol. 28: 232-236 (1990), whereas four antigenically different activities were distinguished by Kamp and Van Leengoed, J. Clin. Microbiol. 27: 1187-1191 (1989). Whether such activities are functions of one or more molecules is not known.

Vaccines proposed thus far for preventing infections by Actinobacillus pleuropneumoniae are mostly based on whole live cells, attenuated cells, lysates, culture supernatants, or extracts of A. pleuropneumoniae. WO-A-80,02113 (or Canadian Patent 1,189,790) teaches a vaccine for controlling pleuropneumonia in pigs, containing A. pleuropneumoniae cells, cell fragments etc. and, as an adjuvant, material derived from Bordetella pertussis. EP-A-420,743 proposes a vaccine containing inactivated toxin of serotype 1 and optionally an inactivated toxin of another serotype of A. pleuropneumoniae; it provides protection against serotype 1 and partial protection against other serotypes. EP-A-354,628 discloses a universal vaccine against A. pleuropneumoniae, which contains extracellular proteins from two different serotypes, and is effective against all A. pleuropneumoniae serotypes. Although these known vaccines provide protection against some or even all of the field strains of A. pleuropneumoniae, the active compounds are not known. As a result, control, verification, and standardisation of vaccines is difficult, since the ratio between active components cannot be optimized and inactive and sometimes adverse components are always present in the vaccines.

The present invention, in one aspect, provides a vaccine for the prevention and/or the treatment of infection by Actinobacillus pleuropneumoniae containing at least an immunogenic part of at least one polypeptide selected from the group consisting of cytolytic proteins of A. pleuropneumoniae produced by recombinant DNA technology, and detoxified derivatives thereof.

It has been found according to the invention that Actinobacillus pleuropneumoniae produces three hemolytic and/or cytotoxic proteins (toxins), hereinafter referred to as cytolytic proteins: Cytolysins I, II and III (ClyI, ClyII and ClyIII). Where the term "Cytolysin" (Cly) is used in the present specification, this shall thus be understood to comprise any extracellular protein produced by any strain of A. pleuropneumoniae and producing any adverse effect (be it hemolytic, cytotoxic or other or both) on cells or tissues of an infected animal; where appropriate it shall be understood also to comprise immunogenically active parts of these proteins or derivatives thereof having diminished adverse effects. Protection against infections by any of the known serotypes of A. pleuropneumoniae is conferred to an animal by administering an effective amount of all three cytolysins, and partial or complete protection against specific serotypes is conferred by administering one or two of the cytolysins, depending on the serotype or serotypes in question.

Thus, the vaccine of the invention contains at least one of the three cytolysins I, II and III, preferably two, and more preferably three. The cytolysins may be present in the vaccine as the naturally occurring proteins, or they may be present as derivatives containing at least an immunogenic part of the proteins, or as a detoxified equivalent. Detoxification shall be understood to mean that the toxic activity of the proteins has been removed to a sufficient degree or for a sufficient number of the protein molecules to provide a vaccine which does not produce an unacceptable toxic reaction in the producing host and/or in the vaccinated animal, whereas it provides a sufficient immune response. Detoxification can be brought about by chemical, physical or enzymatic treatment of the proteins or by substitution, insertion or deletion of one or more nucleotides in the cytolysin genes resulting in the substitution, insertion or deletion of one or more amino acids in the protein. Detoxification can also be achieved by expression of the toxin gene in the absence of the activator gene.

It was found that the cytolysins are encoded by operons wherein the structural toxin gene is flanked at the 5' end by a gene encoding a peptide required to activate the toxin, hereinafter referred to as the activator protein. The cytolysins may be present in the vaccine in the activated or non-activated form.

The cytolysins or their derivatives present in the vaccine are preferably obtained by expression of recombinant DNA encoding the proteins mentioned above. The detoxified cytolysins constitute a further embodiment of the present invention.

In another aspect of the invention a process for producing a cytolytic protein of Actinobacillus pleuropneumoniae or an immunogenic and/or detoxified derivative thereof is provided, which process comprises the steps of: immunogenic part of said cytolytic protein (toxin) optionally including an activator protein, or a derivative thereof; an expression vector; secreting said cytolytic protein, with the vector obtained in step b); sequence(s) of step a);

In yet another aspect, the invention is concerned with a process of producing a vaccine wherein at least one, and preferably two, and more preferably three, of the cytolysins or immunogenic parts thereof, thus produced, are combined with an immunologically acceptable carrier and optionally a suitable adjuvant.

The host cell referred to in the process of producing the cytolysins or their derivatives may be a microorganism, preferably a non-pathogenic microorganism capable of expressing at least one nucleotide sequence encoding the cytolysins by having a strong promoter inducing high expression levels or by allowing the introduction of an exogenous promoter system to induce such high expression levels. A suitable host cell is Escherichia coli.

In a further aspect, the invention provides a nucleotide sequence encoding at least an immunogenic part of a polypeptide selected from cytolytic proteins of Actinobacillus pleuropneumoniae optionally including activator proteins and transport proteins, the latter ones being proteins that assist in the secretion of the cytolytic proteins to the periplasma or the medium. The invention also relates to a system that expresses and secretes said nucleotide sequence and to a vector containing at least one of said nucleotide sequences each one preferably operatively linked to a promoter and optionally an enhancer.

In yet another aspect the invention relates to a host cell containing at least one nucleotide sequence encoding the cytolytic proteins or their derivatives, and capable of expressing them, the nucleotide sequence(s) either being contained as such or as said vector and being either present in the host cell in the genome of the host or as a plasmid. Preferably, the host cell contains nucleotide sequences encoding at least two of the cytolysins, and more preferably it contains the sequences encoding all three cytolysins. The host cell is preferably derived from E. coli.

The invention also provides a vaccine for prophylaxis and therapy of infections by A. pleuropneumoniae containing a microorganism carrying one or more nucleotide sequences encoding at least an immunogenic part of at least one cytolytic proteins of A. pleuropneumoniae or a detoxified derivative thereof. The microorganism may be an attenuated microorganism such as an attenuated virus or a bacterium. Administration of the vaccine results in multiplication of the microorganism and thus in production of the immunogen.

The invention further relates to diagnostic means for detecting infection by A. pleuropneumoniae. Specifically, the invention is concerned with an antibody, preferably a monoclonal antibody, raised against one of the native cytolysins and useful as a component of a diagnostic kit for detecting infection by A. pleuropneumoniae; antibodies raised against modified cytolysins are useful for determining protection by these modified cotylysins. Antibodies raised against native or modified cytolysins can also be used for passive immunisation of infected animals.

In another aspect, the invention provides a DNA-probe comprising at least a part of a nucleotide sequence encoding a cytolysin of Actinobacillus pleuropneumoniae which may be used in a diagnostic method and a diagnostic kit for detecting infection by A. pleuropneumoniae. Another method of diagnosing an A. pleuropneumoniae infection is to determine the presence of A. pleuropneumoniae cytolysins in a subject whereby protein pattern is indicative of the infective serotype or group of serotypes.

In the appended drawings, which form a part of the present disclosure,

FIGS. 1/A-1/F shows the nucleotide sequence of the cytolysin I gene and its activator gene of Actinobacillus pleuropneumoniae serotype 9 (reference strain CVI 13261) and the corresponding sequence of amino acid residues (SEQ ID NO:1:);

FIGS. 2/A-2/D shows the nucleotide sequence of the cytolysin II gene and its activator and transport genes of Actinobacillus pleuropneumoniae strain serotype 9 (reference strain CVI 13261), and the corresponding sequence of amino acid residues (SEQ ID NO:2:);

FIGS. 3/A-3/C shows the preliminary nucleotide sequence of the cytolysin III gene of Actinobacillus pleuropneumoniae serotype 8 (reference strain CVI 405), and the corresponding sequence of amino acid residues (SEQ ID NO:3:);

FIG. 4 schematically shows a RTX-toxin operon comprising the toxin gene (A), the activator gene (C), and the transporter genes (B, D) as well as the operation of the gene products thereof;

FIGS. 5a-d shows the clyIICA determinant of A. pleuropneumoniae serotype 9 and PCR amplification products;

FIG. 6 schematically shows the amplification and cloning of the clyIICA 3' flanking sequence by inverse PCR;

FIG. 7 shows the ClyII determinant organization of the A. pleuropneumoniae serotypes 1-12;

FIG. 8 shows the ClyI determinant organization of the A. pleuropneumoniae serotypes 1-12; and

FIG. 9 illustrates the expression and secretion of cytolysins I, II, and III in recombinant E. coli.

According to the invention it has been found that pathogenesis of Actinobacillus pleuropneumoniae infections can be attributed to three extracellular proteins. These proteins have approximate molecular weights of 105,000, 103,000, and 120,000 respectively. The 105,000 and 103,000 dalton proteins are immunologically related to each other. These proteins were found to be excellent tools for providing protection of animals, in particular pigs, against A. pleuropneumoniae infections of any serotype. Although factors that were thought to be responsible for the pathogenicity of A. pleuropneumoniae were referred to in the prior art as hemolysins and cytotoxins, it has been found now that both cytotoxic and hemolytic activities can result from a single molecule, and hence these proteins are denoted herein as cytolysins (Cly's): the 105 kDa protein as ClyI, the 103 kDa protein as ClyII, and the 120 kDa protein as ClyIII. The nucleotide sequence of the clyI and clyII genes and the preliminary sequence of clyIII is given in FIGS. 1, 2 and 3, respectively.

In FIG. 1 the amino acid sequences of the ClyI C protein (activator), ClyI A protein (cytolytic protein ClyI), ClyI B protein (transport protein), and ClyI D protein (transport protein) are indicated below the nucleotide sequence.

In FIG. 2 the amino acid sequences of the ClyII C protein (activator) and ClyII A protein (cytolytic protein ClyII), are indicated below the nucleotide sequence.

In FIG. 3 the amino acid sequence of the ClyIII A protein (cytolytic protein ClyII) is indicated below the nucleotide sequence.

For the cloning and characterization of the genetic determinants for these proteins three different screening techniques were used: hybridization with an lkt DNA probe, selection for hemolytic activity, and reacion with monoclonal antibodies. On the basis of the reaction pattern with a set of MAbs it was concluded that ClyII is responsible for what has been described by others as HlyII activity (Frey, J., and J. Nicolet (1990) J. Clin. Microb. 28: 232-236). ClyI is identical to HlyI. Since we found no differences between the ClyII amino acid sequence of serotype 9 and that of an RTX toxin identified in serotype 5, the latter also must be responsible for HlyII activity and not for HlyI as has been suggested by others (Chang, Y. et al. (1989) DNA, 8: 635-647; MacInnes, J. I. et al. (1990) J. Bacteriol. 172: 4587-4592). For ClyII we now have shown, for the first time, that both a (weak) hemolytic activity as well as a (moderate) cytotoxic activity are clearly confined in a single protein.

ClyI, ClyII and ClyIII are members of the RTX cytotoxin family. This finding is not only based on immunological data but also on the similarities between primary sequences, hydropathy profiles and the secretion of active toxin by the hlyBD genes of E. coli. The sequenced areas of the ClyI, ClyII and ClyIII encoding operons possess all the general characteristics of other RTX toxin operons (cf. Strathdee, C. A., and R. Y. C. Lo. (1989) J. Bacteriol. 171: 916-928).

With respect to the genetic organization of the ClyII operon we found a striking difference with other RTX operons. The ClyII operon does not contain secretion genes contiguous to the toxin gene. Sequence alignment studies suggested that in an ancestral clyII operon a recombination event occured at position 3490 thereby disrupting the ancestral clyIIB gene. Intact secretion genes are, however, present elsewhere in the genomes of A. pleuropneumoniae serotypes. These secretion genes, however, belong to intact (serotypes 1, 5, 9, 10, 11) or disrupted (serotypes 2, 4, 7, 8, 12) ClyI operons. This is based on sequence data and on the observation that a 7.4 kb NsiI/HindIII DNA fragment covering the clyB gene and approximately 4.5 kb of upstream sequences of serotype 9 encodes a 105 kDa protein indistinguishable from ClyI. This means that the ancestral clyIIBD genes have been lost from the serotype 9 genome. In addition these data indicate that secretion of both ClyI and ClyII is dependent on only a single set of secretion genes. Since these secretion genes belong to the ClyI operon, these genes are referred to herein as clyIBD. Three extra nucleotides are present in front of clyIBD in a region which forms a rho-independent transcription termination signal in other RTX determinants (cf. Strathdee, C. A., and R. Y. C. Lo. (1989) J. Bacteriol. 171: 5955-5962). Furthermore the row of seven T residues which is present in these signals has been changed in clyI to the sequence TTATTT. These nucleotide changes might affect the efficiency of transcription termination or the regulation of this process leading to another level of clyBD expression.

The finding that the primary amino acid sequence of the serotype 9 ClyII is completely identical to the serotype 5 hemolysin and also the finding that (almost) completely identical clyIICA genes are present in serotypes 1, 2, 3, 4, 7, 8, 11 and 12, suggests an important role for ClyII in pathogenesis. The observation that ClyII is produced in all serotypes except serotype 10 and that ClyII is the only extracellular cytolysin of serotypes 6, 7, and 12, supports this view.

The ClyII determinant of the reference strains of all twelve A. pleuropneumoniae serotypes were studied and it was demonstrated, by southern hybridization, that clyIICA sequences are present in all serotypes, except 10. This is in agreement with the observation that serotype 10 is the only serotype not secreting ClyII. PCR amplification of the clyICA sequences of the serotypes carrying these genes resulted in equally sized products for all serotypes, except 6. The clyIICA genes of the serotypes 1, 2, 3, 4, 5, 7, 8, 9, 11 and 12, giving equally sized PCR fragments, were compared by extensive RFLP studies. For these studies we used four different restriction enzymes, which together have 57 recognition sites in the clyIICA sequence of serotype 9 and are therefore very suitable for a detailed comparative study. These studies showed very similar restriction patterns of clyIICA for the ten serotypes examined. These results give strong evidence that the clyIICA genes of the serotypes 1, 2, 3, 4, 5, 7, 8, 9, 11 and 12 have a very similar primary structure. Only three differences among the clyIICA genes of the 10 serotypes were found in these RFLP studies, and this low number is illustrative for the high degree of similarity between the clyII genes. Compared to the serotype 9 sequence additional sites were found for Sau3AI in serotype 5 at position -94, and for RsaI in serotype 7 close to position 2818 or 3143. Furthermore a small deletion between position 510 and 690 was found in serotype 8 by HinfII digestion. Sequence comparison of the serotype 5 and 9 clyIICA sequences showed this additional Sau3AI site in serotype 5. From this comparison it was also expected that in serotype 5 an additional HpaII site at position 209, a three basepair deletion at position 51 and a single base-pair deletion at position 44 would be present. No evidence was found either for the additional HpaII site, when analysing the clyIICA fragments of serotype 5 and 9, after digestion with this enzyme, or for the deletions when analyzing the sizes of the restriction fragments generated by AluI, HinfII, RsaI or Sau3AI. The absence of these sequence differences shows that the serotype 5 and 9 clyIICA sequences are even more similar to each other than expected from the published DNA sequences.

Intact transporter genes, clyIIBD, contiguous with the clyIICA genes were not found among the twelve serotypes. Hybridization of the proposed clyIBD sequences of serotype 9 to genomic DNA of the twelve serotypes showed hybridization to all serotypes, excluding 3 and 6. This indicates that all serotypes, but 3 and 6, do contain the clyIBD transporter genes. The translation products of these genes may act in trans and account for the transmembrane transport of ClyII. The transporter proteins for ClyII of serotypes 3 and 6 however remain to be identified. To our knowledge the proposed complementation of the RTX transporter genes of two RTX operons is the first evidence that these transporter genes are exchangeable in a naturally occurring organism.

The fact that most serotypes secrete ClyII, and that serotype 7 and 12 secrete ClyII as the only cytolysin illustrates the role of this toxin in porcine pleuropneumonia. Immunization with ClyII will induce antibodies directed against ClyII of all serotypes. Furthermore, the very similar clyIICA genes may be the targets of choice for diagnosis of A. pleuropneumoniae infection, since their sequences are present and highly similar in all serotypes, except serotype 10. There is good evidence that field strains of most, if not all, serotypes produce the same cytolytic activities as the reference strains.

Table A shows the extracellular protein pattern and their hemolytic and cytotoxic activity for the various serotypes of Actinobacillus pleuropneumoniae. Table B shows the same protein and activity pattern wherein the immunologically related serotypes are grouped together. ______________________________________ Serotype 1     2     3   4   5   6   7   8   9   10  11 12 ______________________________________ 120 kDa =      .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. ClyIII 105 kDa = ClyI .rect-solid.    .rect-solid.    .rect-solid. .rect-solid. .rect-solid. 103 kDa = .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-soli d. .rect-solid. .rect-solid. .rect-solid. .rect-solid.  .rect-solid. .rect-solid. ClyII Hemolytic S W W W S N W W S S S W Cytotoxic S S S S S N M S S S S M ______________________________________ S = strong activity; M = moderate activity; W = weak activity; N = none .rect-solid. = protein band is present ______________________________________ Serotype   1     5     9   11  2   3   4   8   7   12  10 ______________________________________ 120 kDa = ClyIII               .rect-solid. .rect-solid. .rect-solid. .rect-solid. 105 kDa = ClyI .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. 103 kDa = ClyII .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. .rect-solid. Hemolytic S S S S W W W W W W S Cytotoxic S S S S S S S S M M ______________________________________ S S = strong activity; M = moderate activity; W = weak activity; N = none .rect-solid. = protein band is present

A vaccine containing ClyII or an immunogenic part thereof or a detoxified derivative thereof will provide protection against infections by Actinobacillus pleuropneumoniae serotypes 7 and 12, whereas it might provide partial protection against other serotypes except 10. Similarly, a vaccine containing ClyI or an effective part or derivative thereof will provide protection against serotype 10 and partial protection against serotypes 1, 5, 9 and 11, whereas a vaccine containing ClyIII or an effective part or derivative thereof will provide partial protection against serotypes 2, 3, 4, 6 and 8. Further a vaccine containing ClyII and ClyI or effective parts or derivatives thereof will provide protection against infection by serotypes 1, 5, 7, 9, 10, 11 and 12, and partial protection against the other serotypes; a vaccine containing ClyII and ClyIII or effective parts or derivatives thereof will provide protection against infection by serotypes 2, 3, 4, 6, 7, 8 and 12, and partial protection against the other serotypes except 10; a vaccine containing ClyI and ClyIII or effective parts or derivatives thereof provide protection against infection by serotypes 10, and partial protection against the other serotypes except 7 and 12. A preferred form of the vaccine contains ClyI, ClyII and ClyIII or immogenic parts or detoxified derivatives thereof, and is effective against all known and probably also against any still unknown serotype of A. pleuropneumoniae.

The vaccine of the invention contains the polypeptide or polypeptide derivatives in immunogenically effective amounts, for example between 0.1 and 1000 .mu.g, more particularly between 1 and 100 .mu.g of protein per dosage unit. An important advantage of the invention is that both the absolute and the relative amounts of the immunogens can be adjusted according to the intended use. In contrast, all prior art vaccines contain immunogenic factors in fixed ratios, since they were produced by live A. pleuropneumoniae cells, and separation of the factors was not contemplated and hardly possible. The optimum levels and ratios depend on the nature of the infection against which protect is required, the characteristics of the animals to be protected and other factors known to the skilled person. The vaccine may be administered in a conventional way, such as intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally or orally.

In addition to the cytolysin or part or derivative thereof, the vaccine may comprise an immunologically acceptable carrier, such as aqueous diluents, suspending aids, buffers; furthermore, excipients and adjuvants known in the art may be present. Suitable adjuvants include aluminum hydroxide, Freund's adjuvant (complete or incomplete), bacteria such as Bordetella pertussis or E. coli or bacterium derived matter, immune stimulating complex (iscom), oil, saponin, oligopeptides or other adjuvants known to those skilled in the art. The protein may also be coupled to an acceptable carrier molecule, particularly a natural or synthetic polymer such as polypeptides, polysaccharides, polystyrene, etc. The vaccine may also contain other immunogens related to other diseases in a prophylactically or therapeutically effective amount, to obtain a multivalent vaccine.

The cytolysin or part or derivative thereof may also be fused to another polypeptide; such other polypeptide may be a carrier polypeptide or, advantageously, a second and possibly a third cytolysin or part or derivative thereof. In a preferred embodiment, the vaccine contains a fused polypeptide comprising immunogenic parts of two or three cytolysins. Such a fused polypeptide may be prepared by coupling of the relevant polypeptides, or by fusing the nucleotide sequences encoding said polypeptides followed by suitable expression of the fused nucleotide sequence.

In the process of producing a cytolytic protein of A. pleuropneumoniae or a part or a derivative thereof, suitable for use in the vaccine as described above, in step a) a nucleotide sequence encoding a cytolysin is selected and optionally modified by insertion, substitution or deletion of nucleotides to obtain a sequence encoding an immunogenically active and/or a detoxified protein. The selection of the nucleotide sequence may be performed by screening the gene library of A. pleuropneumoniae using established methods, as illustrated in the examples to the present specification. The nucleotide sequence may then be cloned and isolated; alternatively, the nucleotide sequence may be synthesized. The sequence preferably comprises the sequence encoding an activator protein for the cytolysin, which may be the activator of the cytolysin itself; in the latter case for example, the nucleotide sequence may comprise the clyIICA gene.

The nucleotide sequence is then inserted in a suitable vector in step b). Such a vector may or may not comprise a promoter and optionally an enhancer. The promoter can be selcted to obtain the desired level of expression. Modification of the nucleotide sequence may be performed in the vector, instead of before insertion as explained above. Suitable vectors are art-known.

Step c) can be carried out using standard techniques (see for example: Maniatis, T. et al, (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory). The host cell in which the vector containing the nucleotide sequence is transferred preferably also produces transport proteins allowing the cytolysin or part or derivative thereof to pass the host cell membrane and even be screted into the medium, and thus to be easily harvested. The transport proteins may be derived from A. pleuropneumoniae or from the host cell or from another organism. The host cell is advantageously E. coli.

The nucleotide sequence to be used for producing the cytolysin can be derived from the sequence shown in FIGS. 1, 2 and 3, relating to the clyI (SEQ ID NO: 1:), clyII (SEQ ID NO:2:), and clyIII (SEQ ID NO:3:) genes respectively. The nucleotide sequence can comprise the entire gene, or part thereof encoding at least an epitope of the protein. The nucleotide sequence can be modified by deletions, substitutions or insertions, in particular those which result in a sequence encoding a detoxified derivative of the cytolysin, or those which result in a sequence which, although modified, still encodes the amino acid sequence of the cytolysin or derivative thereof.

Another advantageous type of vaccine provided by the present invention is a vaccine which does not contain the immunogenic protein or proteins as described above, but which contains a recombinant expression system such as a microorganism, carrying a nucleotide sequence encoding said immunogenic protein, for example integrated in its genome or present as an expression vector. Immunization is then induced by administration of the vaccine containing this expression system and subsequent replication and expression in the vaccinated animal. Examples of microorganisms that car be used for this purpose include bacteria such as Salmonella or E. coli, bacteriophages, and viruses, such as vaccinia virus, adenovirus, baculovirus, SV40 retrovirus, hepatitis B virus and pseudorabies virus; other examples are cells which have been transformed with one of these viruses or with other vectors and cells wherein these viruses replicate. These recombinant expression systems constitute an aspect of the present invention.

Monoclonal antibodies to the cytolysins or immunogenic parts or derivatives thereof may be produced in a known manner, e.g. by immunizing a suitable animal with the cytolysin or an appropriate epitope thereof, fusing the resulting cells producing the antibody to the cytolysin with myeloma cells, selecting and cloning the resulting hybridoma cells to produce the antibody. The antibodies to cytolysin I, II and III or to parts of these proteins can be used in a diagnostic method for assaying an infection with A. pleuropneumoniae. The antibodies may be employed in an agglutination assay wherein the antibody may be coupled to a solid particle. The antibodies may be labeled by an enzyme, a luminescent substance, a radioactive isotope, a complexing agent, or by other known means; they may be used in a sandwich assay with a second antibody, one of the two being labeled. The antibodies may be a part of a diagnostic kit, which further contains conventional components for carrying out an immunoassay.

The antibodies are also useful as a means of passive immunization of an animal against A. pleuropneumoniae wherein the antibody inhibits the activity of cytolysins that are introduced by infection. A vaccine to be used for this purpose comprises antibodies to one or more, preferably three different, cytolysins, optionally together with suitable carriers and adjuvants.

The nucleotide sequences illustrated in FIGS. 1, 2 and 3 or in particular suitable parts thereof are also useful as diagnostic tools. Such DNA probes can be used for determining the presence of A. pleuropneumoniae in biological samples of animals. The DNA probes of the invention are used according to known techniques for sampling, hybridization, possible amplification and detection. The DNA probes can be part of a diagnostic kit, which may further contain usual components, such as filters, labeling substances, diluents, amplification or detection aids, etc.

Materials and Methods:

Bacterial strains, plasmids and cloning vectors.

The reference strain CVI 13261 of A. pleuropneumoniae serotype 9 was used as DNA source. The gene library was made in bacteriophage lambda Gem11 (Promega) and propagated in E. coli LE 392 (Sambrook, J., E. F. Fritsch, and T. Maniatis. (1989). Molecular cloning. A laboratory manual. Second edition. Cold Spring Harbor Laboratory Press. Cold Spring Harbor. N.Y.). Specific DNA fragments were (sub)cloned in pKUN plasmid vectors (Konings, R. N. H., et al. (1987) Methods Enzymol. 153: 12-34) and transformed into E. coli JM101 (Sambrook, supra). Plasmid pLKT 52, containing the RTX determinant of P. haemolytica, was prepared by Dr. R. Lo (Strathdee, C. A., and R. Y. C. Lo. (1989) J. Bacteriol. 171: 916-928). Plasmid pLG575, a pACYC184 based plasmid containing the hlyB and hlyD secretion genes of E. coli was prepared by Dr. C. Hughes (Gygi, D., C. Hughes, et al (1990) Mol. Microbiol. 4: 123-128).

Construction and screening of the DNA library.

High molecular weight DNA was isolated by SDS/proteinase K lysis, phenol and phenol/chloroform extractions, and precipitation with ethanol (Sambrook, supra). With this DNA a library was constructed in lambda Gem11 according to the methods recommended by the supplier of the vector arms (Promega). Plaque lifts from this library were hybridized with appropriate restriction fragments which were labelled in advance with [.sup.32 P]dATP (Amersham) using the nick translation kit of Boehringer, Mannheim. Plaques that hybridized were visualized by exposure to X-ray film (Eastman Kodak). The library was also screened for the presence of hemolytic plaques. For that purpose plaques, grown at 37.degree. C. on a Luria broth agarplate, were overlaid with 0.8% agarose containing 5% sheep erythrocytes, 10% Serum Plus (Hazelton) and 0.5 times Eagle's minimal essential medium (Flow laboratories, Irvin, England) in phosphate buffered saline. The plates were incubated at 37.degree. C. for 6 to 12 hr. Selected plaques were purified to homogenicity by at least two cycles of plating and screening.

DNA manipulation and sequence analysis.

DNAs were digested to completion with restriction enzymes according to the specifications of the enzyme supplier (Pharmacia LKB, Sweden). The resulting fragments were separated by electrophoresis on 0.8% agarose gels. Desired fragments were electrophoretically eluted from gel slices and further purified by extractions with phenol and chloroform and precipitation with ethanol. Fragments were (sub)cloned in pKUN plasmid vectors (Konings, supra) by standard molecular biological techniques (Sambrook, supra). Progressive unidirectional deletions were made with the Erase-a-base system from Promega. Nucleotide sequences were determined by the dideoxy chain termination method (Sanger, F. et al (1977) Proc. Natl. Acad. Sci. USA. 74: 5463-5467). The sequences were analyzed using the PCGENE (Intel ligenetics Corp., Mountain View, Calif.) and Wisconsin GCG (University of Wisconsin) analysis software packages.

Gene Screen Plus nylon membranes (Du Pont NEN) were used for Southern blot analysis. The blots were hybridized with DNA probes, labelled as described above, according to the instructions of the membrane supplier. Before exposure the blots were washed a final time with 0.1.times. SSC, 0.1% SDS for 30 min at 65.degree. C. for homologous probes and with 1.times. SSC, 0.1% SDS for 30 min at 50.degree. C. for heterologous probes. All other DNA manipulations were done with standard molecular biological techniques (Sambrook, supra).

Immunoblotting, monoclonal antibodies (MAbs) and toxin bioassays.

Proteins present in recombinant plaques, cells or supernatants of stationary growth cultures were electrophoresed through reducing and denaturing 6% polyacrylamide gels (Laemmli, U. K. (1970) Nature (London) 227: 680-685). The separated proteins were stained with silver or blotted onto nitrocellulose with a semidry blotting apparatus (Bio-rad Laboratories Inc.). The blots were incubated according to the method of Towbin (Towbin, H., et al (1979) Proc. Natl. Acad. Sci. USA. 76: 4350-4354) with convalescent swine serum derived from an A. pleuropneumoniae serotype 9 infected pig or with MAbs specific for ClyI and/or ClyII. MAb CVI-ApCly 9.1 and 9.2 recognize ClyI, MAb CVI-ApCly 9.3 ClyII, and MAb CVI-ApCly 9.4 reacts with ClyI and ClyII (see example 3). Bound antibodies were detected with an anti mouse or anti swine immunoglobulin G-alkaline phosphatase conjugate (Zymed Laboratories Inc.) and color development with the substrates nitroblue tetrazolium (Merck) and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim).

Recombinant toxin, isolated from logarithmic growing cultures, was tested for hemolytic and cytotoxic activity as described earlier (Kamp, E. M., and L. A. M. G. van Leengoed (1989) J. Clin. Microbiol. 27: 1187-1191). Hemolytic and cytotoxic titers were expressed as the reciprocal of the highest dilution showing at least 50% lysis of the target cells.

Gene cloning.

To determine whether A. pleuropneumoniae serotype 9 encoded for toxins related to the RTX cytotoxin family, a 3.7 kilobasepairs (kb) PvuI/SalI DNA fragment derived from the leukotoxin (lkt) determinant of P. haemolytica (Stathdee, C. A. and R. Y. C. Lo. (1987) Infect. Immun. 55: 3233-3236) and containing lktA, the 3'-end of lktC and the 5'-end of lktB (lktCAB) was hybridized to genomic DNA. Three specific DNA fragments were found to be homologous to the probe. The lktCAB probe was then used to screen a library of the A. pleuropneumoniae serotype 9 DNA which was prepared in the vector lambda Gem11. Forty recombinants reacted as strongly positive. To determine whether recombinants with cytolytic activity but without any detectable homology to lkt DNA existed, the library was also screened for the presence of recombinants capable of hemolysis of sheep red blood cells. Three recombinant plaques showed clear hemolytic activity. These hemolytic clones hybridized however with the lktCAB probe, indicating that they shared identical sequences with the clones that were found to be positive with the lkt probe. The hemolytic clones expressed a 103 kDa protein that was absent in non-hemolytic clones. This 103 kDa protein reacted with MAbs specific for ClyII and not with MAbs specific for ClyI (see below). These data indicated that we had cloned the ClyII gene.

To localize the ClyII gene in the 9-21 kb long inserts of the selected recombinants, we digested the DNA of 23 positive clones, including the hemolytic ones, with the restriction enzyme HindIII. The resulting fragments were electrophoresed, blotted onto nylon membranes and hybridized with the lktCAB probe. All recombinants contained a 2.4 kb fragment homologous to the probe. Several recombinants also contained a 4.4 kb fragment that hybridized. Others contained a hybridizing fragment of variable length in addition to the 2.4 kb fragment. Apparently only a part of the 4.4 kb HindIII fragment is present in the latter clones and has been ligated to one of the vector arms. These data provided a location for the ClyII gene (clyIIA).

Although the lktCAB probe used for screening contained approximately 300 bp of the lktB secretion gene, it appeared that none of the 9-21 kb inserts of the selected clones contained intact B and/or D genes. To investigate whether such sequences were present elsewhere in the genome, a 1.2 and a 0.7 kb EcoRV DNA fragment, covering both the 3' end of the lktB gene and the 5' end of the lktD gene of P. haemolytica (lktBD, 24), were hybridized with genomic DNA. A 4.3 kb HindIII fragment hybridized. This fragment was absent from the three hemolytic clones and all the clones that were selected with the lktCAB probe. From these data we concluded that the genome of A. pleuropneumoniae does contain sequences related to the RTX B and D secretion genes but that these sequences are not contiguous to the ClyII toxin gene.

In order to clone the RTX B and D related DNA, HindIII digested and size fractionated genomic DNA of strain CVI 13261 was ligated into a HindIII digested pKUN plasmid. After transformation into E. coli and colony hybridization with lktBD we were able to isolate a clone that contained the 4.3 kb HindIII fragment. Using this fragment we also isolated a 7.0 kb BglII/EcoRV fragment that overlapped the 4.3 kb HindIII fragment at the 5'-end, and a 4.2 kb BamHI fragment that overlapped the 4.3 kb HindIII fragment at the 3'-end. Restriction analysis and Southern hybridization provided a location for the postulated secretion genes clyBD.

Nucleotide sequence analysis.

The clyIICA locus and the clyBD locus were subjected to nucleotide sequence analysis. The established sequences and the derived amino acid sequences of the major open reading frames are shown in FIG. 2. Both loci contained two major open reading frames; these were named clyIIC, clyIIA, clyB and clyD (see also FIG. 1 and FIG. 2). The maps of restriction sites deduced from the sequences correlated well with the maps of restriction sites as determined from the cloned DNA and the genomic DNA (data not shown). Thus no detectable rearrangements had occurred during the cloning procedure. The sequences were numbered starting at -231 (clyIICA locus) and -592 (clyBD locus) to correspond to the orientation and location of the major open reading frames. In clyIICA the open reading frame from 1 to 477 (clyIIC) codes for a polypeptide of 159 amino acids (18.5 kDa) and the frame from 519 to 3386 (clyIIA) for a polypeptide of 956 amino acids (102.5 kDa). The latter protein is the ClyII toxin and, as other RTX toxins, contains glycine rich repeats near the carboxy terminus. In clyBD the frame from 1 to 2133 (clyB) codes for a polypeptide of 711 amino acids (80.2 kDa) and the frame from 2142 to 3575 (clyD) for a polypeptide of 478 amino acids (54.9 kDa).

These protein sequences were very similar to the protein sequences of the RTX determinants of E. coli, P. haemolytica and A. pleuropneumoniae serotype 5. Their mutual hydropathy profiles (Kyte, J. and R. Doolittle (1982) J. Mol. Biol. 157: 105-132) were also quite similar. The ClyIIC and ClyIIA proteins were more homologous to the LktC and LktA proteins of P. haemolytica than to the HlyC and HlyA proteins of E. coli (Stathdee, C. A. and R. Y. C. Lo (1987) Infect. Immun. 55: 3233-3236). In addition the ClyIIA toxin was identical to the 105 kDa toxin identified by Chang et al. in serotype 5 (Chang, Y. et al (1989) DNA 8: 635-647). The ClyIIC protein differed however from its counterpart in serotype 5 at three positions; at amino acid position 5 (extra residue), between residues 41 and 47 (frameshift due to an insertion and deletion of nucleotide residues at positions 125 and 138) and around amino acid position 65 (nucleotide sequence of TGGGCC in serotype 9 and TCCCGG in serotype 5).

The sequence of clyIICA was highly homologous to that of other RTX sequences up to position 3490. This position corresponded to amino acid residue 12/13 of known RTX B secretion proteins. Instead of RTX B protein related sequences we found an open reading frame in the opposite DNA strand downstream this position. This finding confirmed that in serotype 9 no RTX-B related sequences were contiguous with the toxin gene. Probably a recombination occured at position 3490 (amino acid position 12/13 of the truncated RTX-B homolgue) in the clyII operon.

Identification and secretion of ClyII.

A 2.7 kb DNA fragment extending from the 5' end of the insert of one of the selected recombinants up to the KpnI site downstream clyIIA was ligated into pUC18 DNA. E. coli cells that contained this plasmid produced a 103 kDa protein. This protein reacted with a convalescent swine serum, with MAbs specific for ClyII, and not with MAbs specific for ClyI. To provide additional evidence that clyIIA encodes for the 103 kDa ClyII, we electrophoresed the proteins present in clyIICA containing E. coli cells and the proteins present in culture supernatants of serotype 9 alongside a mixture of both preparations. The data clearly indicated that the clyIIA encoded protein comigrates with ClyII.

To assess whether the ClyII toxin also shared functional relatedness with the enterobacterial RTX cytolysins, E. coli cells carrying the clyIICA genes were cotransformed with a compatible plasmid coding for the E. coli hlyBD secretion proteins. The intra- and extracellular proteins of these cells and also of cells that contained either one of these plasmids were assayed for the presence of ClyII. ClyII was only secreted from the cells when the secretion genes were present in trans. These data therefore demonstrated hlyBD mediated export of ClyII across E. coli membranes and a functional relationship between ClyII and the RTX toxin family.

To study the biological activity of ClyII, culture supernatants and cell lysates of the same set of cells were tested for hemolytic and cytotoxic activity. The cytolytic activities in these supernatants and cell extracts perfectly matched with the presence of the ClyII protein among these preparations. These data also indicated that ClyII had two activities: a moderate cytotoxic activity and a weak hemolytic activity. These activities are schematically represented in Tables A and B.

Identification of ClyI.

A 7.4 kb NsiI/HindIII DNA fragment containing the clyB gene and approximately 4.5 kbp of upstream sequences (FIG. 1) was ligated into pUC18 DNA. The proteins produced by cells that contained this plasmid were electrophoresed in parallel with culture supernatant of A. pleuropneumoniae serotype 9 and of ClyII secreting E. coli cells. After blotting we screened for the presence of ClyII, ClyI and ClyII+ClyI. The data demonstrated that the 7.4 kb NsiI/HindIII fragment encodes a 105 kDa protein which is indistinguishable from ClyI and which is clearly different from ClyII. This ClyI protein could also be secreted from E. coli cells when they contained the hlyBD secretion genes in trans. From these data we concluded that the clyBD genes form part of an RTX operon that codes for ClyI. Since the secretion genes belong to the ClyI operon, these genes are now referred to as clyIBD. The upstream sequence of clyIBD containing the clyICA genes was sequenced as described before. The sequence is shown FIG. 1. The genomic organization of the ClyI determinant was determined for the 12 serotypes of Actinobacillus pleuropneumoniae and is depicted in FIG. 8.

Cloning of the gene encoding ClyIII

Genomic DNA of Actinobacillus pleuropneumoniae serotype 8 was partially digested with the restiction enzyme Sau3A to fragments with an average size of about 1000 basepairs. These fragments were partially filled in using Klenow DNA polymerase and dGTP and dATP. The plasmid expression vector pUEX2 (Bressan, G. M. and K. K. Stanley (1987) Nucl. Acid Res. 15: 10056) was digested with the restriction enzyme SalI and partially filled in using Klenow DNA polymerase and DCTP and dTTP. The modified fragments were ligated into the linearized vector and E. coli strain LE392 was transformed with this ligation mixture. Approximately 90,000 independent recombinants were grown at 37.degree. C. and after two hours of induction of the synthesis of .beta.-galactosidase fusion proteins at 42.degree. C., the proteins present in the recombinants were bound to nitrocellulose membranes. The membranes were screened with MAb 2.2 (see example 3), and immunoreactive clones were visualized using rabbit anti mouse serum conjugated with alkaline phosphatase. Three immunoreactive clones were found, 3.4, 5.4, and 7.4. Clones 3.4 and 7.4 contained a 400 base-pair fragment of A. pleuropneumoniae serotype 8, clone 5.4. contained a 1000 bp fragment. Since these fragments cross-hybridized, they contained similar DNA sequences. Sequence analysis of one of these fragments demonstrated that it did not contain the complete clyIII gene. To obtain the complete clyIII gene, genomic DNA of A. pleuropneumoniae serotype 8 was digested to completion with the restriction enzyme HindIII. The resulting fragments were separated on a 0.75% agarose gel and after transfer to nitrocellulose they were hybridized with the DNA fragment present in clone 7.4 which had been labeled with .sup.32 P. A 3200 bp fragment hybridized. This fragment was eluted from the gel and cloned into HindIII restricted plasmid pGEM7Z(+) (Promega) by standard molecular biological techniques. One of the resulting clones, clone 5.2, was shown to harbor the 3200 bp fragment. The nucleotide sequence of this fragment was determined and analysis of the seuence revealed an open reading frame of a distal part of a gene coding for a protein homologous to the E. coli .alpha.-hemolysin, and the proximal part of a gene coding for a protein homologous to Hly B of E. coli. It was concluded that the 3200 bp fragment of clone 5.2 comprises sequences of an RTX-toxin operon and hence that ClyIII is a member of the RTX-toxin family. Thus clone 5.2 contained the distal part of an RTX A-gene (toxin gene) and the proximal part of an RTX B-gene (coding for a transport proten). The full length sequence of the putative clyIII gene was obtained by the cloning and sequencing of a 4200 bp NsiI/XbaI fragment (clone 6.1) that hybridized with a 1200 bp HindIII/XbaI fragment of clone 5.2 and overlapped with the 5'-end of the fragment in clone 5.2. The nucleotide sequence showed the open reading frame of a gene coding for the proximal part of a RTX A protein and a complete RTX C protein.

For expression purposes we constructed a plasmid that contained an XbaI/XhoI fragment made by combining the 4200 bp NsiI/XbaI insert of clone 6.1 with a 1300 bp XbaI/XhoI fragment of clone 5.2. E. coli cells that contained this plasmid produced a protein with a molecular weight of about 120,000 dalton that reacted with the ClyIII specific MAb 2.2. This demonstrated that we had cloned the gene encoding ClyIII.

Cotransformation of these cells with plasmid pLG575, carrying the Hly B and D transport proteins of the E. coli hemolysin determinant, resulted in the secretion of the 120,000 dalton protein. The secreted protein had a strong cytotoxic activity for porcine lung macrophages. It did not show any hemolytic activity to sheep erythrocytes.

In conclusion, the 120,000 dalton protein is demonstrated to be the ClyIII protein since it has the same size, the same immunological properties, and the same biological activity as the ClyIII protein of A. pleuropneumoniae. Furthermore from hybridization studies we know that sequences homologous to the ClyIII coding gene are only present in the serotypes 2, 3, 4, and 8, the only serotypes that produce ClyIII.

The nucleotide sequence of the ClyIII gene was determined essentially as described above. The sequence is shown in FIG. 3. pleuropneumoniae Serotypes

Materials and methods

Bacterial strains, genomic DNA, plasmids and oligonucleotides.

The reference strains for the twelve serotypes of A. pleuropneumoniae were used as source of genomic DNA. The reference strains for the serotypes 1 to 12 were respectively s4047, 1536, 1421, M62, K17, Femo, WF83, 405, 13261, D13039, 56153 and 8329. High molecular weight DNA was isolated by proteinase K/SDS lysis, phenol/chloroform extractions and precipitation with ethanol (Maniatis, T. et al (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory). DNA fragments were cloned with the plasmid pGEM7Zf(+) (Promega Corporation, Madison, Wis.) in E. coli strain JM101, using standard molecular biology techniques (Maniatis, supra). Sequences of oligonucleotides used for the PCR are given. Their position in serotype 9 clyIICA is indicated between brackets. Position 1 is the first base of the clyIIC reading frame (see Example 1).

Underlined sequences are recognition sites for the restriction enzymes HindIII in oligonucleotide 322 and NotI in 284 and 285. These sites were introduced for cloning purposes (NotI was not used in this study) by the modification of one (322), two (285) or three (284) bases of the original serotype 9 clyIICA sequence.

Southern blots and dot-blots.

Southern blots of restriction fragments of genomic DNA, separated on 0.8% agarose gel and dot-blots of high molecular weight genomic DNA were made with Genescreen plus membranes (NEN Research Products, Boston, Mass.). For the Southern blots 1 .mu.g of DNA per lane was used, and for the dotblots 50 ng per dot. The blots were hybridized overnight in a Hybaid hybridization oven at 65.degree. C. DNA with .alpha..sup.32 P dCTP (Amersham, UK) labeled DNA prepared by random prime labeling (Random Primed DNA labeling kit, Boehringer Mannheim, Mannheim, FRG). The blots were washed with a final stringency of 0.2 SSC (1.times. SSC is 0.15 M sodium chloride, 0.015 M sodium citrate) and 1% Sodium Dodecyl Sulphate (SDS) for 15 min at 65.degree. C. Radioactivity was detected by autoradiography using intensifying screens on X-omat AR film (Eastman Kodak, Rochester, N.Y.).

Amplification and radiolabeling of DNA by the PCR.

The clyIICA sequences were amplified by the PCR using genomic DNA from the A. pleuropneumoniae reference strains as a template. The PCR was done in a volume of 50 ml containing approximately 100 ng template DNA, 1 .mu.M of each of two specific oligonucleotides, 1 U Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn.), 0.2 mM of each of four deoxynucleotide triphosphates (dNTP), 25 mM Tris/HCl, pH 8.7, 2.5 mM MgCl.sub.2 and 0.05% gelatin. The reaction mixture was covered with a drop of mineral oil to prevent evaporation and subjected to 30 PCR cycles of 1 min at 92.degree. C., 1 min at 55.degree. C. and 3 min at 74.degree. C. in a Thermal Cycler (Perkin Elmer Cetus). Amplified DNA fragments were separated by agarose gel electrophoresis and extracted from the gel with GeneClean (Bio 101 LaJolla, Calif). Two to four nanogrammes of the purified DNA fragments were radiolabeled in a successive PCR with a dNTP concentration of 0.05 mM each, and 5 mCi .alpha..sup.32 P dCTP. Unincorporated dNTPs were removed by precipitating the radiolabeled DNA from the phenol/chloroform extracted reaction mixture with ethanol in the presence of 2.5 M ammonium acetate. The relatively high concentration of unlabeled dNTPs used in this PCR will decrease the specific activity of the synthesized DNA, but will favor the fidelity and complete extension of the PCR products, desirable for RFLP studies (Jansen, R. and F. D. Ledley (1989) Gene Anal. Techn. 6: 79-83).

Inverse PCR, cloning and sequence analysis.

Inverse PCR (Ochman, H. et al (1988) Genetics 120: 621-625) was done under the same conditions as the PCR described above, except that the extension reaction was 90 sec at 74.degree. C. The template DNA for the inverse PCR was prepared as follows: HindIII digested genomic DNA was size fractionated by agarose gel electrophoresis and extracted from the gel with GeneClean. These fragments were circularized by self-ligation with T4 ligase in a volume of 50 .mu.l containing approximately 100 ng of DNA fragments. One tenth of the ligation product was used as a template in the inverse PCR. The inverse PCR resulted in a high background of aspecific products. The desired amplification products were size fractionated by agarose gel electrophoreses and extracted from the gel with GeneClean. Reamplification of these fragments was done in a subsequent PCR using the same oligonucleotides and reaction conditions. The resulting fragments were cloned into pGEM7Zf(+), using the HindIII site resulting from the circularization reaction and an artificial HindIII site in oligonucleotide 322. Sequence analysis of the cloned fragments was done with the T7 sequencing kit (Pharmacia, Uppsala, Sweden) by using oligonucleotides specific for the SP6 and T7 promotors (Promega).

Restriction fragment analysis.

Radiolabeled DNA fragments were separately digested with the restriction enzymes AluI, Sau3AI, RsaI and HinfII. The resulting DNA fragments were separated on a vertical 5% polyacrylamide (acryl:bisacryl is 19:1) gel with dimensions of 400.times.500.times.1.5 mm, buffered with 0.18 M Tris/boric acid (pH 7.8), 0.5 mM EDTA (TBE). The digestion products were visualized by autoradiography of the dried gel.

Presence of clyIICA in the serotypes 1-12 and comparison of their sequences. The presence of clyIICA sequences in the genomic DNA of the twelve A. pleuropneumoniae serotypes was detected by using dot-blot hybridization with serotype 9 clyIICA sequences as a probe. This probe, comprising bases 315 to 3233, hybridized to the DNA of all serotypes, except serotype 10. Genomic DNA of the twelve serotypes was subjected to PCR using four oligonucleotides derived from the serotype 9 clyIICA genes. FIG. 5A shows the position of these oligomers in the two contiguous genes. Set 283/284 was used for amplification of the 5' region, 285/286 for the 3' region and 283/286 for amplification of full length clyIICA. The PCR on the DNA of the serotypes 1, 2, 3, 4, 5, 7, 8, 11 and 12 resulted for each primer set in amplification products of the same size as obtained with serotype 9 DNA (1750 bp for set 283/284, 2050 bp for set 285/286 and 3200 bp for the set 283/286). The 3.2 kb full length fragments of the serotypes 1,2,3,4,5,7,8,11 and 12, generated by using set 283/286, showed identical restriction maps for the enzymes HindIII, Xba I and Pst I as serotype 9 clyIICA. Serotype 6 gave an identically sized amplification product as serotype 9 for set 285/286, but amplification products which were approximately 1800 bp longer for the sets 283/284 and 283/286. Serotype 10 did not give visible amplification products using either set of oligonucleotides.

The degree of similarity between the clyIICA genes of the serotypes 1, 2, 3, 4, 5, 7, 8, 9, 11, and 12 was studied by RFLP analysis of the full length clyIICA sequences, generated in the PCR with the oligonucleotide set 283/286. The DNA fragments were labeled with .alpha..sup.32 P dCTP and digested with the restriction enzymes AluI, HinfII, RsaI or Sau3AI. The obtained restriction fragments were analyzed by gel electrophoresis and autoradiography. For each of the four restriction enzymes, the number and sizes of the DNA fragments obtained from clyIICA of the serotypes 1, 2, 3, 4, 5, 7, 8, 9, 11, and 12 appeared to be very similar. The RFLP studies on the serotype 12 clyIICA sequences were done in separate experiments.

Cloning and analysis of the sequences adjacent to clyIIA.

The proximal part of a putative clyIIB gene was found adjacent to clyIIA of serotype 5 and 9 (see example 1). In serotype 5 this sequence extended to at least 108 bases, but in serotype 9 it was truncated after 37 bases. To study the presence of this putative clyIIB gene in the other serotypes we cloned the sequences distal of clyIIA 3' by inverse PCR as outlined in FIG. 6. We chose HindIII digestion of the genomic DNA, since the genomic HindIII fragments of most serotypes containing these sequences have a workable size for inverse PCR amplification, and an unique HindIII site is present in clyIICA of all serotypes at base 2008. The probe used in this analysis comprised bases 2008 to 3493 of serotype 9 clyIICA. Among the twelve serotypes, four differently sized HindIII fragments hybridized to this probe. A 2.8 kb HindIII fragment in the serotypes 2, 3, 5, 7, and 8, a 2.3 kb fragment in the serotypes 1, 9, 11, and 12, a 10 kb fragment in serotype 4 and a 4.3 kb fragment in serotype 6. Inverse PCR using these HindIII fragments is expected to result in amplification products, approximately 1300 bases smaller (the number of bases between the oligonucleotides 158 and 322) than the hybridizing HindIII fragments. The inverse PCR resulted in the desired amplification products for all serotypes, except 4 and 6. The failure to get amplification products for serotypes 4 and 6 was probably due to the large sizes of the HindIII fragments, exceding the range of inverse PCR amplification in this system. We cloned the inverse PCR products of the serotypes 1, 2, 3, 5, 7, 8, 9, 11, and 12 into pGEM7Zf(+) and determined their nucleotide sequences. All these serotypes appeared to have a truncated clyIIB adjacent of clyIIA, and two different truncation points were identified, at base 37 and at base 501. We identified two major types of sequences downstream of clyIIB. Type I, present in the serotypes 1, 7, 9, 11, and 12, and type II present in the serotypes 2, 3, 5, and 8. A subtype of type I was identified in serotype 7 and 12, since both had seven additional bases, AACCACT, at position 3664. The sequence of type I was illustrated in example 1 as part (base 3490 to 4499) of the serotype 9 clyIICA sequence. The protein sequence derived from the 501 bases truncated clyIIB has a similarity of 71% to the serotype 9 clyIB (14), and 64% to the P. haemolytica lktB. The type I and II sequences did not show any similarity with each other or with RTX-CABD sequences. FIG. 7 shows a schematic presentation of the genomic structure of the clyIICA and the truncated clyIIB genes of the twelve serotypes. The type I sequences are represented by hatched boxes and type II sequences by dotted boxes. None of the twelve serotypes contained a clyII operon with intact genes for the B and D transporter proteins (see also example 1). Hybridization experiments demonstrated however the presence of clyIBD sequences in all serotypes except 3. pleuropneumoniae by Monoclonal Antibodies

Materials and Methods

Bacterial strains.

The sources and designations of the reference strains for A. pleuropneumoniae serotypes 1 to 12 aere those mentioned in example 2. The field strain CVI 12946 was isolated in the Netherlands from the lung of a pig that died from pleuropneumonia. This strain was typed as serotype 2 by slide agglutination (Kamp, E. M. et al (1987) Vet. Microbiol. 13: 249-257).

Preparation of culture filtrates.

Actinobacillus pleuropneumoniae strains were cultured in Eagle minimal essential medium plus Earle salts (Flow Laboratories, Irving, UK) and 10% Serum Plus (Hazelton Research Products, Lexena, Kans.) as described earlier (Kamp E. M. and A. M. G. van Leengoed (1989) J. Clin. Microbiol. 27 1187-1191). Cultures were centrifuged for 30 min at 10,000.times. g, and the supernatants were sterilized by passing them through membrane filters of 0.2 .mu.m pore size (Gelman Sciences Inc., Ann Arbor, Mich.). Culture filtrates were stored in aliquots at -20.degree. C. until further use.

Swine sera.

Specific-pathogen-free 4-week-old pigs were endo-bronchially inoculated with 103 colony forming units of A. pleuropneumoniae 1536 (serotype 2) or 13261 (serotype 9). Blood samples were collected 2 months after inoculation. Blood was allowed to clot overnight at 4.degree. C., serum was collected and heated for 60 min at 60.degree. C. to inactivate complement. Sera were stored in aliquots at -20.degree. C.

Monoclonal antibodies.

Culture filtrates of strains CVI 12946 (serotype 2) and CVI 13261 (serotype 9) were detoxified with 0.5% formalin and used to immunize Balb/c mice. The immunization schedule and the preparation of hybridoma cell lines were as described in detail by van Zijderveld et al. (Infect. Immun. 57: 1192-1199). Hybridomas were tested for antibody in enzyme-linked immunosorbent assays (ELISA) using microtiter plates coated with culture medium or culture filtrates of strains CVI 12946 or CVI 13261. Hybridomas that tested positive with a culture filtrate and negative with the culture medium were cloned twice by limiting dilution.

The resulting monoclonal cell lines were used to produce ascites fluid in pristane-primed Balb/c mice. Antibody was purified from the ascites fluid by precipitation with 40% ammonium sulfate and dialysis against phosphate-buffered saline, pH 7.2 (PBS). The MAbs were stored in aliquots containing 8 mg protein/ml at -20.degree. C. The immunoglobulin isotype of the MAbs was determined in immuno- diffusion tests using mouse isotype-specific antisera (Nordic, Tilburg, The Netherlands).

ELISAs.

The procedures for ELISA were as described in detail by Van Zijderveld et al. (supra). We used polystyrene microdilution plates coated with culture filtrates of either strain CVI 12946 or CVI 13261. The optimal dilutions for coating were determined by checkerboard titrations using the swine sera as positive sera. Coated plates were stored at -20.degree. C.

Titers of the MAb preparations were determined in an indirect ELISA. Bound antibodies were detected with peroxidase labeled anti-mouse immunoglobulins (Dakopatts, Copenhagen, Denmark) and hydrogen peroxide mixed with 5-aminosalicylic acid. Titers were expressed as the logarithm of the reciprocal of the highest dilution giving an A.sub.450 of 50% of the maximum obtainable absorbance value.

A competition ELISA was used to determine whether the MAbs recognized different epitopes. MAbs were conjugated to horseradish peroxidase (Boehringer, Mannheim, Federal Republic of Germany). Serial two-fold dilutions of 50 ml samples of non-conjugated MAbs were incubated in coated microdilution plates for 30 min at 37.degree. C. Plates were not washed, and 50 ml of the optimal dilution of each of the peroxidase-conjugated MAbs was added per well. Plates were further incubated for 1 h, washed, and then incubated with the substrate.

Hemolysin assay.

Serial five-fold dilutions of 1 ml of the culture filtrates were tested for hemolytic activity as described by Frey and Nicolet (1988, FEMS Microbiol. Lett. 55: 41-46); a suspension of 1% sheep erythrocytes in Tris-buffered saline, pH 7.2, with 10 mM CaCl2 was used. Just before the determination of the A.sub.540, 20 .mu.l 0.1 N HCl was added to each tube to change the color of the phenol red in the medium to yellow. Hemolytic activities were expressed in hemolytic units; one hemolytic unit was defined as the absorbance value of a solution of 1 part of the 1% erythrocytes suspension and 3 parts distilled water.

Hemolysin inhibition assays.

Inhibition of hemolytic activity was tested in two assays. The first assay was as described by Frey and Nicolet (supra); serial two-fold dilutions of 1 ml samples of all culture filtrates were incubated for 1 h at 37.degree. C. with 10 .mu.l samples of one of the MAbs, swine sera, or buffer. Then, 1 ml of a suspension of 1% sheep erythrocytes in Tris-buffered saline with CaCl.sub.2 was added to each tube and from that point on the test was further performed as the hemolysin assay was. The hemolytic activity of the culture filtrates of serotypes 2, 3, 6, and 8 was too weak to determine inhibition. Therefore, we also tested inhibition of hemolytic activity by inoculating A. pleuropneumoniae serotypes 1 to 12 onto sheep blood agar plates that contained 0.05% nicotinamide adenine dinucleotide and a 1:100 dilution of one of the MAbs or a 1:200 dilution of the swine sera. Plates without antibodies were used as controls. The plates had a diameter of 5.5 cm and contained 5 ml medium each. Per serotype, one colony of an 18 hour old culture was suspended in 1 ml PBS. Very fine capillary tubes were used to inoculate the plates with these suspensions. After incubating the plates overnight at 37.degree. C. in an atmosphere of 5% CO.sub.2, hemolytic zones were measured and compared to those of the controls. Inhibition was expressed as when hemolytic zones were similar to those of the controls, as + when hemolytic zones were present but were more than 50% smaller than the controls, and as + when no hemolytic zones were detected.

Cytotoxin assay.

The isolation of porcine alveolar macrophages and the cytotoxin assay have been described earlier in detail (Kamp, E. M. and L. A. M. G. van Leengoed (1989) J. Clin. Microbiol. 27: 1187-1191).

Cytotoxin inhibition assay.

Serial two-fold dilutions of 50 .mu.l samples of all culture filtrates (except serotype 6) were made in PBS in flat-bottomed microdilution plates (8 rows per serotype). Either PBS (control) or one of the MAbs or polyclonal swine sera were added to each row (50 .mu.l per well). MAbs were used in a dilution of 1:100 and swine sera in a dilution of 1:200. Plates were sealed, shaken, and incubated for 1 h at 37.degree. C. An amount of 50 .mu.l alveolar macrophages was added to each well and from this point on, the test was performed as the cytotoxin assay was. Cytotoxin titers were determined and compared with the titer of the control. Inhibition was expressed as when cytotoxin titers were the same as the titer of the control, as + when titers were two to four-fold lower than the titer of the control, and as + when titers were more than four-fold lower than the titer of the control.

Sodium dodecyl polyacrylamide gel electrophoresis and Western blot analysis. Proteins in the culture filtrates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in a Mini Protean II slab cell according to the recommendations of the manufacturer (Bio-Rad, Richmond, Calif.). We used a 4% stacking gel with a 7.5% separating gel, an acrylamide/bisacrylamide ratio of 19/1, 0.75 mm spacers, and combs with 15 wells. Each well was loaded with 15 .mu.l samples and electrophoresed at 25 V on ice. Proteins were transferred onto nitrocellulose filters electrophoretically according to the recommendations of the manufacturer of the Blot system (Novablot, LKB, Uppsala, Sweden). The blots were probed with a 1:200 dilution of the MAbs or a 1:400 dilution of the swine sera. Bound immunoglobulins were visualized by using peroxidase-labeled goat anti-mouse or goat antiswine immunoglobulins (Dakopatts) and HRP Color Development Reagent (Bio-Rad). Control blots were probed with buffer instead of MAb.

Hemolytic and cytotoxic activities.

Culture filtrates of all serotypes except of serotype 6 were cytotoxic and hemolytic (Table A). Hemolytic activity of culture filtrates of serotypes 2, 3, 4, 7, 8, and 12 were much weaker than those of serotypes 1, 5, 9, 10, and 11. All reference strains of A. pleuropneumoniae serotypes 1 to 12, including serotype 6, were hemolytic on blood agar. The hemolytic zones around colonies of serotypes 2, 3, 4, 7, 8, and 12 were much smaller than the zones around serotypes 1, 5, 9, 10, and 11. Hemolysis of serotype 6 could only be detected after removal of the colony.

Monoclonal antibodies and swine sera.

For this study we selected five MAbs and two swine sera. MAb CVI-Apcly 2.2 was raised against serotype 2 and tested in ELISA positive with serotype 2 and negative with serotype 9. MAbs CVI-Apcly 9.1 and 9.2 were raised against serotype 9 and tested positive with serotype 9 and negative with serotype 2. In contrast, the two swine sera and MAbs CVI-Apcly 9.3 and 9.4, which were also raised against serotype 9, tested positive with both serotype 2 and 9. The MAbs did not block each other in a competition ELISA, indicating that they recognized different epitopes.

Inhibition of hemolytic and cytotoxic activity.

MAbs and swine sera were tested for inhibition of hemolytic and cytotoxic activity of serotypes 1 to 12. MAb CVI-Apcly 9.1 and pig anti serotype 9 serum reduced the hemolytic activity of culture filtrates of serotypes 1, 5, 9, 10, and 11 with 80% or more. MAbs CVI-Apcly 9.3 and 9.4 and the swine sera against serotypes 2 and 9 totally reduced the hemolytic activity of the culture filtrates of serotypes 4, 7, and 12. Because the hemolytic activity of the culture filtrates of serotypes 2, 3, 6, and 8 was too weak to reliably determine inhibition of hemolysis in the liquid assay, we also tested the MAbs and swine sera for their ability to inhibit hemolysis on blood agar. The results of this test were similar to those of the liquid assay.

MAb CVI-Apcly 2.2 was raised against serotype 2 and inhibited cytotoxic activity of serotypes 2, 3, 4, and 8. MAb CVI-Apcly 9.1 was raised against serotype 9 and inhibited the hemolytic activity and the cytotoxic activity of serotypes 1, 5, 9, 10, and 11, suggesting that both activities are functions of the same protein. MAb CVI-Apcly 9.2 was also raised against serotype 9 and inhibited the cytotoxic but not the hemolytic activity of serotypes 1, 5, 9, 10, and 11. In contrast, MAbs CVI-Apcly 9.3 and 9.4, which were also raised against serotype 9, did not inhibit hemolytic or cytotoxic activity of these serotypes. Surprisingly, these two MAbs inhibited the hemolytic activity and cytotoxic activity of serotypes 7 and 12. This suggests that the hemolytic and cytotoxic activity of these serotypes are functions of the same protein. In addition, MAbs CVI-Apcly 9.3 and 9.4 inhibited the hemolytic but not the cytotoxic activity of serotypes 2, 3, 4, and 8.

Pig serum raised against serotype 2 inhibited the hemolytic and cytotoxic activity of serotypes 2, 3, 4, 6, 7, 8, and 12, whereas pig serum raised against serotype 9 inhibited the hemolytic and cytotoxic activity of serotypes 1, 5, 6, 7, 9, 10, 11, and 12 and the hemolytic but not the cytotoxic activity of serotypes 2, 3, 4, and 8.

Western blot analysis.

The selected MAbs and swine sera were used to probe Western blots of the culture filtrates of serotypes 1 to 12. MAb CVI-Apcly 2.2 reacted with a protein of approximately 120 kDa in filtrates of serotypes 2, 3, 4, and 8. MAbs CVI-Apcly 9.1 and 9.2 reacted with a protein of approximately 105 kDa in filtrates of serotypes 1, 5, 9, 10, and 11 and MAb CVI-Apcly 9.3 reacted with a protein also of approximately 105 kDa in filtrates of all serotypes except serotype 10. The reaction of MAb CVIApcly 9.3 with the proteins of serotypes 3, 6, and 8 was very weak and not always visible.

To test whether MAbs CVI-Apcly 2.2, 9.1, 9.2, and 9.3 recognized different proteins, we probed a blot with one MAb, washed the blot thoroughly, and then tested it with another MAb. This procedure was repeated until all four MAbs were tested. Three proteins became visible. One protein of approximately 120 kDa was detected by MAb CVI-Apcly 2.2 in serotypes 2, 3, 4, and 8; a second protein of approximately 105 kDa was detected by MAbs CVI-Apcly 9.1 and 9.2 in serotypes 1, 5, 9, 10, and 11; and a third protein of approximately 103 kDa was detected by MAb CVI-Apcly 9.3 in all serotypes except serotype 10 (Table A).

MAb CVI-Apcly 9.4 reacted with the 105 kDa protein and the 103 kDa protein, indicating that these two proteins have epitopes in common.

Western blot analysis using the swine sera confirmed the distribution of the three proteins among the 12 serotypes. Pig serum raised against serotype 2 recognized a protein of approximately 120 kDa in serotypes 2, 3, 4, and 8 and a protein of approximately 103 kDa in all serotypes except serotype 10. Pig serum raised serotype 9 recognized a protein of approximately 105 kDa in serotypes 1, 5, 9, 10, and 11, and a protein of approximately 103 kDa in all serotypes except serotype 10.

Cells of E. coli strain LE 392 that contained plasmid pLG 575 (Gygi, D. et al. (1990) Mol. Microbiol. 4: 123-128) were transformed with plasmids that contained ClyI, ClyII, or ClyIII encoding genes. These cells were grown at 37.degree. C. in Luria Broth medium, supplemented with the appropriate antibiotics and preferably with 10% Feutal Calf Serum, for about 6-8 h to an optical density at 620 nanometer of approximately 0.5. The culture was centrifuged, the supernatant was sterilized by treatment with a bacterio-staticum and stored. The proteins present in the culture supernatants and that reacted with a mixture of Mabs 9.1, 9.3 and 2.2 are shown in FIG. 9. The purified Cly proteins from those supernatants, or preferably the crude supernatants, are mixed in a predetermined ratio and subsequently mixed with an appropriate adjuvant and used for vaccination.

FIG. 9 is a diagrammatic representation of a Western blot showing expression and secretion of ClyIII (lanes 1-4), ClyII (lanes 5, 6) and ClyI lanes 7,8) by recombinant E. coli cells that contain the cytolysin gene in question together with transport genes of E. coli itself. The proteins were electrophoresed on SDS-PAGE, blotted on nitrocellulose and visualized with MAb 2.2, 9.1 and 9.3. #             SEQUENCE LISTING - -  - - (1) GENERAL INFORMATION: - -    (iii) NUMBER OF SEQUENCES: 3 - -  - - (2) INFORMATION FOR SEQ ID NO: 1: - -      (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8370 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1: - - AGATTAATGA GCGATATTGT TATAAAATCA TAATGTAAAC CTCATTTGTA AT - #GAATTGGT     60 - - AAATTATATA AATAATCAAA AAACTTACTT TTTTTTATTT TTATCGGTAA GT - #ATTTACA     120 - - TCAAGTCAGA CAAACGGCAA TATTGTTATA AATCTGGGGG GATGA   - # 165 - - ATG AGT AAA AAA ATT AAT GGA TTT GAG GTT TT - #A GGA GAG GTG GCA 21 - #0 Met Ser Lys Lys Ile Asn Gly Phe Glu Val Le - #u Gly Glu Val Ala 1               5  - #                 10 - #                 15 - - TGG TTA TGG GCA AGT TCT CCT TTA CAT CGA AA - #G TGG CCG CTT TCT 25 - #5 Trp Leu Trp Ala Ser Ser Pro Leu His Arg Ly - #s Trp Pro Leu Ser 20 - #                 25 - #                 30 - - TTG TTA GCA ATT AAT GTG CTA CCT GCG ATT GA - #G AGT AAT CAA TAT 30 - #0 Leu Leu Ala Ile Asn Val Leu Pro Ala Ile Gl - #u Ser Asn Gln Tyr 35 - #                 40 - #                 45 - - GTT TTG TTA AAG CGT GAC GGT TTT CCT ATT GC - #A TTT TGT AGC TGG 34 - #5 Val Leu Leu Lys Arg Asp Gly Phe Pro Ile Al - #a Phe Cys Ser Trp 50 - #                 55 - #                 60 - - GCA AAT TTG AAT TTG GAA AAT GAA ATT AAA TA - #C CTT GAT GAT GTT 39 - #0 Ala Asn Leu Asn Leu Glu Asn Glu Ile Lys Ty - #r Leu Asp Asp Val 65 - #                 70 - #                 75 - - GCC TCG CTA GTT GCG GAT GAT TGG ACT TCC GG - #C GAT CGT CGA TGG 43 - #5 Ala Ser Leu Val Ala Asp Asp Trp Thr Ser Gl - #y Asp Arg Arg Trp 80 - #                 85 - #                 90 - - TTT ATA GAT TGG ATA GCA CCG TTC GGA GAC AG - #T GCC GCA TTA TAC 48 - #0 Phe Ile Asp Trp Ile Ala Pro Phe Gly Asp Se - #r Ala Ala Leu Tyr 95 - #                100 - #                105 - - AAA CAT ATG CGA GAT AAC TTC CCG AAT GAG CT - #G TTT AGG GCT ATT 52 - #5 Lys His Met Arg Asp Asn Phe Pro Asn Glu Le - #u Phe Arg Ala Ile 110  - #               115  - #               120 - - CGA GTT GAT CCG GAC TCT CGA GTA GGG AAA AT - #T TCA GAA TTT CAT 57 - #0 Arg Val Asp Pro Asp Ser Arg Val Gly Lys Il - #e Ser Glu Phe His 125  - #               130  - #               135 - - GGA GGA AAA ATT GAT AAG AAA CTG GCA AGT AA - #A ATT TTT CAA CAA 61 - #5 Gly Gly Lys Ile Asp Lys Lys Leu Ala Ser Ly - #s Ile Phe Gln Gln 140  - #               145  - #               150 - - TAT CAC TTT GAA TTA ATG AGT GAG CTA AAA AA - #T AAA CAA AAT TTT 66 - #0 Tyr His Phe Glu Leu Met Ser Glu Leu Lys As - #n Lys Gln Asn Phe 155  - #               160  - #               165 - - AAA TTT TCA TTA GTA AAT AGC TAA GGA GAC AA - #C ATG GCT AAC TCT 70 - #5 Lys Phe Ser Leu Val Asn Ser       - #          Met Ala Asn Ser 165  - #                  - #      1 - - CAG CTC GAT AGA GTC AAA GGA TTG ATT GAT TC - #A CTT AAT CAA CAT 75 - #0 Gln Leu Asp Arg Val Lys Gly Leu Ile Asp Se - #r Leu Asn Gln His 5                 - #  10                - #  15 - - ACA AAA AGT GCA GCT AAA TCA GGT GCC GGC GC - #A TTA AAA AAT GGT 79 - #5 Thr Lys Ser Ala Ala Lys Ser Gly Ala Gly Al - #a Leu Lys Asn Gly 20                 - # 25                 - # 30 - - TTG GGA CAG GTG AAG CAA GCA GGG CAG AAA TT - #A ATT TTA TAT ATT 84 - #0 Leu Gly Gln Val Lys Gln Ala Gly Gln Lys Le - #u Ile Leu Tyr Ile 35                 - # 40                 - # 45 - - CCG AAA GAT TAT CAA GCT AGT ACC GGC TCA AG - #T CTT AAT GAT TTA 88 - #5 Pro Lys Asp Tyr Gln Ala Ser Thr Gly Ser Se - #r Leu Asn Asp Leu 50                 - # 55                 - # 60 - - GTG AAA GCG GCG GAG GCT TTA GGG ATC GAA GT - #A CAT CGC TCG GAA 93 - #0 Val Lys Ala Ala Glu Ala Leu Gly Ile Glu Va - #l His Arg Ser Glu 65                 - # 70                 - # 75 - - AAA AAC GGT ACC GCA CTA GCG AAA GAA TTA TT - #C GGT ACA ACG GAA 97 - #5 Lys Asn Gly Thr Ala Leu Ala Lys Glu Leu Ph - #e Gly Thr Thr Glu 80                 - # 85                 - # 90 - - AAA CTA TTA GGT TTC TCG GAA CGA GGC ATC GC - #A TTA TTT GCA CCT 1020 Lys Leu Leu Gly Phe Ser Glu Arg Gly Ile Al - #a Leu Phe Ala Pro 95                 - #100                 - #105 - - CAG TTT GAT AAG TTA CTG AAT AAG AAC CAA AA - #A TTA AGT AAA TCG 1065 Gln Phe Asp Lys Leu Leu Asn Lys Asn Gln Ly - #s Leu Ser Lys Ser 110                 1 - #15                 1 - #20 - - CTC GGC GGT TCA TCG GAA GCA TTA GGA CAA CG - #T TTA AAT AAA ACG 1110 Leu Gly Gly Ser Ser Glu Ala Leu Gly Gln Ar - #g Leu Asn Lys Thr 125                 1 - #30                 1 - #35 - - CAA ACG GCA CTT TCA GCC TTA CAA AGT TTC TT - #A GGT ACG GCT ATT 1155 Gln Thr Ala Leu Ser Ala Leu Gln Ser Phe Le - #u Gly Thr Ala Ile 140                 1 - #45                 1 - #50 - - GCG GGT ATG GAT CTT GAT AGC CTG CTT CGT CG - #C CGT AGA AAC GGT 1200 Ala Gly Met Asp Leu Asp Ser Leu Leu Arg Ar - #g Arg Arg Asn Gly 155                 1 - #60                 1 - #65 - - GAG GAC GTC AGT GGT TCG GAA TTA GCT AAA GC - #A GGT GTG GAT CTA 1245 Glu Asp Val Ser Gly Ser Glu Leu Ala Lys Al - #a Gly Val Asp Leu 170                 1 - #75                 1 - #80 - - GCC GCT CAG TTA GTG GAT AAC ATT GCA AGT GC - #A ACG GGT ACG GTG 1290 Ala Ala Gln Leu Val Asp Asn Ile Ala Ser Al - #a Thr Gly Thr Val 185                 1 - #90                 1 - #95 - - GAT GCG TTT GCC GAA CAA TTA GGT AAA TTG GG - #C AAT GCC TTA TCT 1335 Asp Ala Phe Ala Glu Gln Leu Gly Lys Leu Gl - #y Asn Ala Leu Ser 200                 2 - #05                 2 - #10 - - AAC ACT CGC TTA AGC GGT TTA GCA AGT AAG TT - #A AAT AAC CTT CCA 1380 Asn Thr Arg Leu Ser Gly Leu Ala Ser Lys Le - #u Asn Asn Leu Pro 215                 2 - #20                 2 - #25 - - GAT TTA AGC CTT GCA GGA CCT GGG TTT GAT GC - #C GTA TCA GGT ATC 1425 Asp Leu Ser Leu Ala Gly Pro Gly Phe Asp Al - #a Val Ser Gly Ile 230                 2 - #35                 2 - #40 - - TTA TCT GTT GTT TCG GCT TCA TTC ATT TTA AG - #T AAT AAA GAT GCC 1470 Leu Ser Val Val Ser Ala Ser Phe Ile Leu Se - #r Asn Lys Asp Ala 245                 2 - #50                 2 - #55 - - GAT GCA GGT ACA AAA GCG GCG GCA GGT ATT GA - #A ATC TCA ACT AAA 1515 Asp Ala Gly Thr Lys Ala Ala Ala Gly Ile Gl - #u Ile Ser Thr Lys 260                 2 - #65                 2 - #70 - - ATC TTA GGC AAT ATC GGT AAA GCG GTT TCT CA - #A TAT ATT ATT GCG 1560 Ile Leu Gly Asn Ile Gly Lys Ala Val Ser Gl - #n Tyr Ile Ile Ala 275                 2 - #80                 2 - #85 - - CAA CGT GTG GCG GCA GGC TTA TCC ACA ACT GC - #G GCA ACC GGT GGT 1605 Gln Arg Val Ala Ala Gly Leu Ser Thr Thr Al - #a Ala Thr Gly Gly 290                 2 - #95                 3 - #00 - - TTA ATC GGT TCG GTC GTA GCA TTA GCG ATT AG - #C CCG CTT TCG TTC 1650 Leu Ile Gly Ser Val Val Ala Leu Ala Ile Se - #r Pro Leu Ser Phe 305                 3 - #10                 3 - #15 - - TTA AAT GTT GCG GAT AAG TTT GAA CGT GCG AA - #A CAG CTT GAA CAA 1695 Leu Asn Val Ala Asp Lys Phe Glu Arg Ala Ly - #s Gln Leu Glu Gln 320                 3 - #25                 3 - #30 - - TAT TCG GAG CGC TTT AAA AAG TTC GGT TAT AA - #A GGT GAT AGT TTA 1740 Tyr Ser Glu Arg Phe Lys Lys Phe Gly Tyr Ly - #s Gly Asp Ser Leu 335                 3 - #40                 3 - #45 - - TTA GCT TCA TTC TAC CGT GAA ACC GGT GCG AT - #T GAA GCG GCA TTA 1785 Leu Ala Ser Phe Tyr Arg Glu Thr Gly Ala Il - #e Glu Ala Ala Leu 350                 3 - #55                 3 - #60 - - ACC ACG ATT AAC AGT GTG TTA AGT GCG GCT TC - #C GCA GGT GTT GGG 1830 Thr Thr Ile Asn Ser Val Leu Ser Ala Ala Se - #r Ala Gly Val Gly 365                 3 - #70                 3 - #75 - - GCT GCT GCA ACC GGC TCA TTA GTC GGT GCG CC - #G GTA GCA GCT TTA 1875 Ala Ala Ala Thr Gly Ser Leu Val Gly Ala Pr - #o Val Ala Ala Leu 380                 3 - #85                 3 - #90 - - GTT AGT GCA ATC ACC GGT ATT ATT TCA GGT AT - #T TTA GAT GCT TCT 1920 Val Ser Ala Ile Thr Gly Ile Ile Ser Gly Il - #e Leu Asp Ala Ser 395                 4 - #00                 4 - #05 - - AAA CAG GCA ATC TTC GAA CGA GTT GCA ACG AA - #A TTA GCG AAT AAG 1965 Lys Gln Ala Ile Phe Glu Arg Val Ala Thr Ly - #s Leu Ala Asn Lys 410                 4 - #15                 4 - #20 - - ATT GAC GAA TGG GAG AAA AAA CAC GGT AAA AA - #C TAT TTT GAA AAC 2010 Ile Asp Glu Trp Glu Lys Lys His Gly Lys As - #n Tyr Phe Glu Asn 425                 4 - #30                 4 - #35 - - GGT TAT GAC GCC CGC CAT TCC GCA TTC TTA GA - #A GAT ACC TTT GAA 2055 Gly Tyr Asp Ala Arg His Ser Ala Phe Leu Gl - #u Asp Thr Phe Glu 440                 4 - #45                 4 - #50 - - TTG TTA TCA CAA TAC AAT AAA GAG TAT TCG GT - #A GAG CGT GTC GTT 2100 Leu Leu Ser Gln Tyr Asn Lys Glu Tyr Ser Va - #l Glu Arg Val Val 455                 4 - #60                 4 - #65 - - GCT ATT ACG CAA CAG CGT TGG GAT GTC AAT AT - #C GGT GAA CTT GCC 2145 Ala Ile Thr Gln Gln Arg Trp Asp Val Asn Il - #e Gly Glu Leu Ala 470                 4 - #75                 4 - #80 - - GGC ATT ACT CGC AAA GGT TCT GAT ACG AAA AG - #C GGT AAA GCT TAC 2190 Gly Ile Thr Arg Lys Gly Ser Asp Thr Lys Se - #r Gly Lys Ala Tyr 485                 4 - #90                 4 - #95 - - GTT GAT TTC TTT GAA GAA GGA AAA CTT TTA GA - #G AAA GAA CCG GAT 2235 Val Asp Phe Phe Glu Glu Gly Lys Leu Leu Gl - #u Lys Glu Pro Asp 500                 5 - #05                 5 - #10 - - CGT TTT GAT AAA AAA GTG TTT GAT CCG CTT GA - #A GGT AAA ATC GAC 2280 Arg Phe Asp Lys Lys Val Phe Asp Pro Leu Gl - #u Gly Lys Ile Asp 515                 5 - #20                 5 - #25 - - CTT TCT TCA ATT AAC AAA ACC ACT TTA TTG AA - #A TTT GTT ACC CCG 2325 Leu Ser Ser Ile Asn Lys Thr Thr Leu Leu Ly - #s Phe Val Thr Pro 530                 5 - #35                 5 - #40 - - GTC TTT ACC GCA GGT GAA GAG ATT CGT GAG CG - #T AAG CAA ACC GGT 2370 Val Phe Thr Ala Gly Glu Glu Ile Arg Glu Ar - #g Lys Gln Thr Gly 545                 5 - #50                 5 - #55 - - AAA TAC GAA TAT ATG ACC GAA TTA TTC GTT AA - #A GGT AAA GAA AAA 2415 Lys Tyr Glu Tyr Met Thr Glu Leu Phe Val Ly - #s Gly Lys Glu Lys 560                 5 - #65                 5 - #70 - - TGG GTG GTA ACC GGT GTG CAG TCA CAT AAT GC - #G ATT TAT GAC TAT 2460 Trp Val Val Thr Gly Val Gln Ser His Asn Al - #a Ile Tyr Asp Tyr 575                 5 - #80                 5 - #85 - - ACG AAT CTT ATC CAA TTA GCG ATA GAT AAA AA - #A GGT GAA AAA CGT 2505 Thr Asn Leu Ile Gln Leu Ala Ile Asp Lys Ly - #s Gly Glu Lys Arg 590                 5 - #95                 6 - #00 - - CAA GTG ACC ATT GAA TCT CAT TTG GGT GAG AA - #A AAT GAT CGT ATA 2550 Gln Val Thr Ile Glu Ser His Leu Gly Glu Ly - #s Asn Asp Arg Ile 605                 6 - #10                 6 - #15 - - TAT CTT TCA TCC GGT TCA TCT ATC GTA TAT GC - #G GGT AAC GGA CAT 2595 Tyr Leu Ser Ser Gly Ser Ser Ile Val Tyr Al - #a Gly Asn Gly His 620                 6 - #25                 6 - #30 - - GAT GTA GCA TAT TAC GAT AAA ACC GAT ACA GG - #T TAC TTA ACA TTT 2640 Asp Val Ala Tyr Tyr Asp Lys Thr Asp Thr Gl - #y Tyr Leu Thr Phe 635                 6 - #40                 6 - #45 - - GAC GGA CAA AGT GCA CAG AAA GCC GGT GAA TA - #T ATT GTC ACT AAA 2685 Asp Gly Gln Ser Ala Gln Lys Ala Gly Glu Ty - #r Ile Val Thr Lys 650                 6 - #55                 6 - #60 - - GAA CTT AAA GCT GAT GTA AAA GTT TTA AAA GA - #A GTG GTT AAA ACT 2730 Glu Leu Lys Ala Asp Val Lys Val Leu Lys Gl - #u Val Val Lys Thr 665                 6 - #70                 6 - #75 - - CAG GAT ATT TCA GTT GGA AAA CGC AGT GAA AA - #A TTA GAA TAT CGT 2775 Gln Asp Ile Ser Val Gly Lys Arg Ser Glu Ly - #s Leu Glu Tyr Arg 680                 6 - #85                 6 - #90 - - GAT TAT GAG TTA AGC CCA TTC GAA CTT GGG AA - #C GGT ATC AGA GCT 2820 Asp Tyr Glu Leu Ser Pro Phe Glu Leu Gly As - #n Gly Ile Arg Ala 695                 7 - #00                 7 - #05 - - AAA GAT GAA TTA CAT TCT GTT GAA GAA ATT AT - #C GGT AGT AAT CGT 2865 Lys Asp Glu Leu His Ser Val Glu Glu Ile Il - #e Gly Ser Asn Arg 710                 7 - #15                 7 - #20 - - AAA GAC AAA TTC TTT GGT AGT CGC TTT ACC GA - #T ATT TTC CAT GGT 2910 Lys Asp Lys Phe Phe Gly Ser Arg Phe Thr As - #p Ile Phe His Gly 725                 7 - #30                 7 - #35 - - GCG AAA GGC GAT GAT GAA ATC TAC GGT AAT GA - #C GGC CAC GAT ATC 2955 Ala Lys Gly Asp Asp Glu Ile Tyr Gly Asn As - #p Gly His Asp Ile 740                 7 - #45                 7 - #50 - - TTA TAC GGA GAC GAC GGT AAT GAT GTA ATC CA - #T GGC GGT GAC GGT 3000 Leu Tyr Gly Asp Asp Gly Asn Asp Val Ile Hi - #s Gly Gly Asp Gly 755                 7 - #60                 7 - #65 - - AAC GAC CAT CTT GTT GGT GGT AAC GGA AAC GA - #C CGA TTA ATC GGC 3045 Asn Asp His Leu Val Gly Gly Asn Gly Asn As - #p Arg Leu Ile Gly 770                 7 - #75                 7 - #80 - - GGA AAA GGT AAT AAT TTC CTT AAT GGC GGT GA - #T GGT GAC GAT GAG 3090 Gly Lys Gly Asn Asn Phe Leu Asn Gly Gly As - #p Gly Asp Asp Glu 785                 7 - #90                 7 - #95 - - TTG CAG GTC TTT GAG GGT CAA TAC AAC GTA TT - #A TTA GGT GGT GCG 3135 Leu Gln Val Phe Glu Gly Gln Tyr Asn Val Le - #u Leu Gly Gly Ala 800                 8 - #05                 8 - #10 - - GGT AAT GAC ATT CTG TAT GGC AGC GAT GGT AC - #T AAC TTA TTT GAC 3180 Gly Asn Asp Ile Leu Tyr Gly Ser Asp Gly Th - #r Asn Leu Phe Asp 815                 8 - #20                 8 - #25 - - GGT GGT GTA GGC AAT GAC AAA ATC TAC GGT GG - #T TTA GGT AAG GAT 3225 Gly Gly Val Gly Asn Asp Lys Ile Tyr Gly Gl - #y Leu Gly Lys Asp 830                 8 - #35                 8 - #40 - - ATT TAT CGC TAC AGT AAG GAG TAC GGT CGT CA - #T ATC ATT ATT GAG 3270 Ile Tyr Arg Tyr Ser Lys Glu Tyr Gly Arg Hi - #s Ile Ile Ile Glu 845                 8 - #50                 8 - #55 - - AAA GGC GGT GAT GAT GAT ACG TTA TTG TTA TC - #G GAT CTT AGT TTT 3315 Lys Gly Gly Asp Asp Asp Thr Leu Leu Leu Se - #r Asp Leu Ser Phe 860                 8 - #65                 8 - #70 - - AAA GAT GTA GGA TTT ATC AGA ATC GGT GAT GA - #T CTT CTT GTG AAT 3360 Lys Asp Val Gly Phe Ile Arg Ile Gly Asp As - #p Leu Leu Val Asn 875                 8 - #80                 8 - #85 - - AAA AGA ATC GGA GGA ACA CTG TAT TAC CAT GA - #A GAT TAC AAT GGG 3405 Lys Arg Ile Gly Gly Thr Leu Tyr Tyr His Gl - #u Asp Tyr Asn Gly 890                 8 - #95                 9 - #00 - - AAT GCG CTC ACG ATT AAA GAT TGG TTC AAG GA - #A GGT AAA GAA GGA 3450 Asn Ala Leu Thr Ile Lys Asp Trp Phe Lys Gl - #u Gly Lys Glu Gly 905                 9 - #10                 9 - #15 - - CAA AAT AAT AAA ATT GAA AAA ATC GTT GAT AA - #A GAT GGA GCT TAT 3495 Gln Asn Asn Lys Ile Glu Lys Ile Val Asp Ly - #s Asp Gly Ala Tyr 920                 9 - #25                 9 - #30 - - GTT TTA AGC CAA TAT CTG ACT GAA CTG ACA GC - #T CCT GGA AGA GGT 3540 Val Leu Ser Gln Tyr Leu Thr Glu Leu Thr Al - #a Pro Gly Arg Gly 935                 9 - #40                 9 - #45 - - ATC AAT TAC TTT AAT GGG TTA GAA GAA AAA TT - #G TAT TAT GGA GAA 3585 Ile Asn Tyr Phe Asn Gly Leu Glu Glu Lys Le - #u Tyr Tyr Gly Glu 950                 9 - #55                 9 - #60 - - GGA TAT AAT GCA CTT CCT CAA CTC AGA AAA GA - #T ATT GAA CAA ATC 3630 Gly Tyr Asn Ala Leu Pro Gln Leu Arg Lys As - #p Ile Glu Gln Ile 965                 9 - #70                 9 - #75 - - ATT TCA TCT ACG GGT GCA TTT ACC GGT GAT CA - #C GGA AAA GTA TCT 3675 Ile Ser Ser Thr Gly Ala Phe Thr Gly Asp Hi - #s Gly Lys Val Ser 980                 9 - #85                 9 - #90 - - GTA GGC TCA GGC GGA CCG TTA GTC TAT AAT AA - #C TCA GCT AAC AAT 3720 Val Gly Ser Gly Gly Pro Leu Val Tyr Asn As - #n Ser Ala Asn Asn 995                1000 - #               1005 - - GTA GCA ATT CTT TGA GTTATTCTTT AGCACAAGCA GCTTAAGAT - #A 3765 Val Ala Ile Leu 1010 - - GTTATTTTTA GATGATAAAT AGCAATCCTA TATATATTAG GTGTGTAGGA  - # 3815 - - TTGCTATTTT ATTTATGGAG GAGCAA          - #      ATG GAT TTT TAT - #   3853 - #                  - #        Met Asp Phe Tyr - #                  - #         1 - - CGG GAA GAA GAC TAC GGA TTA TAC GCA CTG AC - #G ATT TTA GCC CAG 3898 Arg Glu Glu Asp Tyr Gly Leu Tyr Ala Leu Th - #r Ile Leu Ala Gln 5                - #  10                - #  15 - - TAC CAT AAT ATT GCT GTA AAT CCG GAA GAA CT - #A AAA CAT AAA TTC 3943 Tyr His Asn Ile Ala Val Asn Pro Glu Glu Le - #u Lys His Lys Phe 20                 - # 25                 - # 30 - - GAC CTT GAA GGA AAA GGC TTA GAT CTA ACC GC - #T TGG CTA TTA GCC 3988 Asp Leu Glu Gly Lys Gly Leu Asp Leu Thr Al - #a Trp Leu Leu Ala 35                 - # 40                 - # 45 - - GCA AAA TCA TTA GAA CTT AAA GCA AAA CAA GT - #A AAA AAA GCG ATT 4033 Ala Lys Ser Leu Glu Leu Lys Ala Lys Gln Va - #l Lys Lys Ala Ile 50                 - # 55                 - # 60 - - GAT CGT TTG GCG TTT ATC GCA CTA CCG GCA CT - #T GTA TGG CGA GAA 4078 Asp Arg Leu Ala Phe Ile Ala Leu Pro Ala Le - #u Val Trp Arg Glu 65                 - # 70                 - # 75 - - GAC GGT AAA CAT TTT ATT TTG ACT AAA ATT GA - #T AAT GAA GCA AAA 4123 Asp Gly Lys His Phe Ile Leu Thr Lys Ile As - #p Asn Glu Ala Lys 80                 - # 85                 - # 90 - - AAA TAT TTA ATT TTT GAT TTG GAA ACG CAT AA - #T CCT CGC ATT TTG 4168 Lys Tyr Leu Ile Phe Asp Leu Glu Thr His As - #n Pro Arg Ile Leu 95                 - #100                 - #105 - - GAA CAA GCG GAA TTC GAG AGC TTA TAC CAA GG - #A AAA CTG ATT TTA 4213 Glu Gln Ala Glu Phe Glu Ser Leu Tyr Gln Gl - #y Lys Leu Ile Leu 110                 1 - #15                 1 - #20 - - GTT GCA TCA AGA GCT TCC ATC GTA GGT AAG CT - #G GCA AAG TTT GAC 4258 Val Ala Ser Arg Ala Ser Ile Val Gly Lys Le - #u Ala Lys Phe Asp 125                 1 - #30                 1 - #35 - - TTC ACT TGG TTT ATA CCG GCG GTA ATT AAG TA - #T CGT AAG ATT TTT 4303 Phe Thr Trp Phe Ile Pro Ala Val Ile Lys Ty - #r Arg Lys Ile Phe 140                 1 - #45                 1 - #50 - - ATT GAA ACG TTA ATT GTT TCA ATT TTT TTG CA - #A ATT TTC GCA CTA 4348 Ile Glu Thr Leu Ile Val Ser Ile Phe Leu Gl - #n Ile Phe Ala Leu 155                 1 - #60                 1 - #65 - - ATT ACA CCG CTT TTT TTC CAA GTC GTG ATG GA - #T AAA GTC TTG GTA 4393 Ile Thr Pro Leu Phe Phe Gln Val Val Met As - #p Lys Val Leu Val 170                 1 - #75                 1 - #80 - - CAC CGA GGT TTT TCA ACC TTA AAT GTG ATT AC - #G GTG GCA TTA GCG 4438 His Arg Gly Phe Ser Thr Leu Asn Val Ile Th - #r Val Ala Leu Ala 185                 1 - #90                 1 - #95 - - ATC GTC GTG CTG TTT GAA ATT GTG CTA AAC GG - #T TTA CGT ACC TAT 4483 Ile Val Val Leu Phe Glu Ile Val Leu Asn Gl - #y Leu Arg Thr Tyr 200                 2 - #05                 2 - #10 - - ATT TTT GCG CAT AGT ACC AGC CGT ATT GAT GT - #G GAG TTG GGA GCA 4528 Ile Phe Ala His Ser Thr Ser Arg Ile Asp Va - #l Glu Leu Gly Ala 215                 2 - #20                 2 - #25 - - AGA TTA TTC AGA CAT TTA TTA GCA CTC CCA AT - #C TCT TAT TTT GAA 4573 Arg Leu Phe Arg His Leu Leu Ala Leu Pro Il - #e Ser Tyr Phe Glu 230                 2 - #35                 2 - #40 - - AAT CGT CGA GTC GGC GAT ACG GTG GCT CGT GT - #A CGA GAA CTC GAT 4618 Asn Arg Arg Val Gly Asp Thr Val Ala Arg Va - #l Arg Glu Leu Asp 245                 2 - #50                 2 - #55 - - CAA ATT CGT AAC TTC TTA ACC GGG CAG GCA CT - #T ACT TCC GTG TTG 4663 Gln Ile Arg Asn Phe Leu Thr Gly Gln Ala Le - #u Thr Ser Val Leu 260                 2 - #65                 2 - #70 - - GAT TTA ATG TTT TCC TTT ATC TTC TTT GCA GT - #G ATG TGG TAT TAC 4708 Asp Leu Met Phe Ser Phe Ile Phe Phe Ala Va - #l Met Trp Tyr Tyr 275                 2 - #80                 2 - #85 - - AGC CCT AAA CTT ACT CTT GTG ATT TTA GGC TC - #G TTA CCG TTT TAT 4753 Ser Pro Lys Leu Thr Leu Val Ile Leu Gly Se - #r Leu Pro Phe Tyr 290                 2 - #95                 3 - #00 - - ATG GGG TGG TCG ATT TTT ATC AGC CCT ATT TT - #A CGT CGC CGT TTA 4798 Met Gly Trp Ser Ile Phe Ile Ser Pro Ile Le - #u Arg Arg Arg Leu 305                 3 - #10                 3 - #15 - - GAT GAA AAA TTC GCA CGT GGT GCG GAC AAT CA - #G TCA TTC TTA GTG 4843 Asp Glu Lys Phe Ala Arg Gly Ala Asp Asn Gl - #n Ser Phe Leu Val 320                 3 - #25                 3 - #30 - - GAA TCG GTG ACT GCA ATC AAT ACG ATT AAA GC - #G TTG GCG GTT ACC 4888 Glu Ser Val Thr Ala Ile Asn Thr Ile Lys Al - #a Leu Ala Val Thr 335                 3 - #40                 3 - #45 - - CCT CAA ATG ACT AAT ACC TGG GAT AAG CAA TT - #A GCC AGC TAT GTA 4933 Pro Gln Met Thr Asn Thr Trp Asp Lys Gln Le - #u Ala Ser Tyr Val 350                 3 - #55                 3 - #60 - - TCG GCG GGA TTC CGT GTA ACC ACA TTA GCT AC - #T ATC GGA CAG CAA 4978 Ser Ala Gly Phe Arg Val Thr Thr Leu Ala Th - #r Ile Gly Gln Gln 365                 3 - #70                 3 - #75 - - GGT GTA CAA TTT ATT CAA AAA GTC GTG ATG GT - #T ATT ACC TTA TGG 5023 Gly Val Gln Phe Ile Gln Lys Val Val Met Va - #l Ile Thr Leu Trp 380                 3 - #85                 3 - #90 - - CTA GGC GCA CAT TTA GTG ATT TCA GGC GAT TT - #A AGT ATC GGA CAA 5068 Leu Gly Ala His Leu Val Ile Ser Gly Asp Le - #u Ser Ile Gly Gln 395                 4 - #00                 4 - #05 - - TTA ATC GCA TTT AAT ATG TTA TCC GGT CAA GT - #G ATT GCA CCG GTG 5113 Leu Ile Ala Phe Asn Met Leu Ser Gly Gln Va - #l Ile Ala Pro Val 410                 4 - #15                 4 - #20 - - ATT CGT TTA GCG CAA CTT TGG CAA GAT TTC CA - #A CAA GTG GGA ATT 5158 Ile Arg Leu Ala Gln Leu Trp Gln Asp Phe Gl - #n Gln Val Gly Ile 425                 4 - #30                 4 - #35 - - TCG GTA ACG CGT TTA GGT GAT GTT TTA AAC TC - #T CCG ACC GAG AGC 5203 Ser Val Thr Arg Leu Gly Asp Val Leu Asn Se - #r Pro Thr Glu Ser 440                 4 - #45                 4 - #50 - - TAT CAA GGA AAA TTG GCG TTA CCG GAA ATT AA - #A GGC GAT ATT ACC 5248 Tyr Gln Gly Lys Leu Ala Leu Pro Glu Ile Ly - #s Gly Asp Ile Thr 455                 4 - #60                 3 - #65 - - TTC CGT AAT ATA CGC TTC CGC TAC AAA CCG GA - #T GCG CCG GTG ATT 5293 Phe Arg Asn Ile Arg Phe Arg Tyr Lys Pro As - #p Ala Pro Val Ile 470                 4 - #75                 4 - #80 - - TTA AAT GAT GTG AAT TTA TCG ATT CAG CAA GG - #T GAA GTG ATC GGT 5338 Leu Asn Asp Val Asn Leu Ser Ile Gln Gln Gl - #y Glu Val Ile Gly 485                 4 - #90                 4 - #95 - - ATC GTA GGA CGT TCA GGC TCA GGG AAG AGC AC - #C TTA ACG AAA TTA 5383 Ile Val Gly Arg Ser Gly Ser Gly Lys Ser Th - #r Leu Thr Lys Leu 500                 5 - #05                 5 - #10 - - ATT CAA CGT TTT TAT ATT CCG GAA AAC GGT CA - #G GTA TTA ATA GAT 5428 Ile Gln Arg Phe Tyr Ile Pro Glu Asn Gly Gl - #n Val Leu Ile Asp 515                 5 - #20                 5 - #25 - - GGG CAT GAT TTA GCA TTG GCG GAT CCG AAC TG - #G CTA CGT CGT CAA 5473 Gly His Asp Leu Ala Leu Ala Asp Pro Asn Tr - #p Leu Arg Arg Gln 530                 5 - #35                 5 - #40 - - GTC GGG GTG GTA TTA CAA GAT AAC GTA CTA TT - #A AAT CGT AGT ATT 5518 Val Gly Val Val Leu Gln Asp Asn Val Leu Le - #u Asn Arg Ser Ile 545                 5 - #50                 5 - #55 - - CGA GAT AAT ATT GCC TTA GCG GAT CCG GGT AT - #G CCA ATG GAA AAA 5563 Arg Asp Asn Ile Ala Leu Ala Asp Pro Gly Me - #t Pro Met Glu Lys 560                 5 - #65                 5 - #70 - - ATT GTC CAT GCG GCA AAA TTA GCC GGC GCA CA - #T GAA TTT ATT TCT 5608 Ile Val His Ala Ala Lys Leu Ala Gly Ala Hi - #s Glu Phe Ile Ser 575                 5 - #80                 5 - #85 - - GAA TTG CGT GAG GGA TAT AAC ACG ATT GTT GG - #T GAG CAA GGT GCG 5653 Glu Leu Arg Glu Gly Tyr Asn Thr Ile Val Gl - #y Glu Gln Gly Ala 590                 5 - #95                 6 - #00 - - GGG CTA TCT GGC GGG CAA CGC CAA CGT ATT GC - #G ATT GCA CGC GCT 5698 Gly Leu Ser Gly Gly Gln Arg Gln Arg Ile Al - #a Ile Ala Arg Ala 605                 6 - #10                 6 - #15 - - TTG GTG AAT AAC CCG AAA ATC TTA ATT TTT GA - #T GAA GCG ACC AGC 5743 Leu Val Asn Asn Pro Lys Ile Leu Ile Phe As - #p Glu Ala Thr Ser 620                 6 - #25                 6 - #30 - - GCA TTA GAT TAT GAA TCC GAG CAT ATC ATC AT - #G CGC AAT ATG CAC 5788 Ala Leu Asp Tyr Glu Ser Glu His Ile Ile Me - #t Arg Asn Met His 635                 6 - #40                 6 - #45 - - CAG ATT TGT AAA GGG AGA ACG GTA ATT ATC AT - #T GCA CAC CGT TTA 5833 Gln Ile Cys Lys Gly Arg Thr Val Ile Ile Il - #e Ala His Arg Leu 650                 6 - #55                 6 - #60 - - TCT ACG GTA AAA AAT GCC GAC CGT ATT ATT GT - #G ATG GAA AAA GGT 5878 Ser Thr Val Lys Asn Ala Asp Arg Ile Ile Va - #l Met Glu Lys Gly 665                 6 - #70                 6 - #75 - - CAG ATT GTG GAA CAA GGT AAG CAT AAA GAG CT - #G CTT GCT GAT CCA 5923 Gln Ile Val Glu Gln Gly Lys His Lys Glu Le - #u Leu Ala Asp Pro 680                 6 - #85                 6 - #90 - - AAC GGC TTA TAT CAC TAC TTA CAC CAA TTA CA - #A TCG GAA TAG GAGGA 5970 Asn Gly Leu Tyr His Tyr Leu His Gln Leu Gl - #n Ser Glu 695                 7 - #00                 7 - #05 - - CTT ATG AAA ACT TGG CTA ATG GGT TTA TAT GA - #G TTT TTC CAA CGC 6015 Met Lys Thr Trp Leu Met Gly Leu - #Tyr Glu Phe Phe Gln Arg 1             - #  5                - #   10 - - TAT AAA ACG GTT TGG ACG GAG ATC TGG AAA AT - #T CGT CAT CAA TTG 6060 Tyr Lys Thr Val Trp Thr Glu Ile Trp Lys Il - #e Arg His Gln Leu 15                 - # 20                 - # 25 - - GAT ACG CCG GAT CGA GAA AAG GAT GAA AAT GA - #A TTT TTA CCT GCA 6105 Asp Thr Pro Asp Arg Glu Lys Asp Glu Asn Gl - #u Phe Leu Pro Ala 30                 - # 35                 - # 40 - - CAC TTA GAG CTG ATT GAA ACA CCG GTG TCA AA - #A AAA CCG AGA TTG 6150 His Leu Glu Leu Ile Glu Thr Pro Val Ser Ly - #s Lys Pro Arg Leu 45                 - # 50                 - # 55 - - ATC GCT TAT TTA ATT ATG CTG TTC CTA TTT TT - #G GCA TTA GTT ATT 6195 Ile Ala Tyr Leu Ile Met Leu Phe Leu Phe Le - #u Ala Leu Val Ile 60                 - # 65                 - # 70 - - TCA ATT GTC AGT CAC GTA GAA ATT GTG GCG AC - #C GCA ACG GGT AAA 6240 Ser Ile Val Ser His Val Glu Ile Val Ala Th - #r Ala Thr Gly Lys 75                 - # 80                 - # 85 - - TTA GCG TTT AGC GAC CGT AGC AAA GAA ATT AA - #G CCG ATT GAA AAC 6285 Leu Ala Phe Ser Asp Arg Ser Lys Glu Ile Ly - #s Pro Ile Glu Asn 90                 - # 95                 - #100 - - GCC TTG GTT AAA GAA ATC TTT GTG CAA GAC GG - #A CAA TTT GTT GAG 6330 Ala Leu Val Lys Glu Ile Phe Val Gln Asp Gl - #y Gln Phe Val Glu 105                 1 - #10                 1 - #15 - - AAA GAT CAG TTG CTG TTA CAC TTG ACC GCA TT - #G GGA GCC GAT GCG 6375 Lys Asp Gln Leu Leu Leu His Leu Thr Ala Le - #u Gly Ala Asp Ala 120                 1 - #25                 1 - #30 - - GAT CAA CAA AAA ACC AAA AGT TCG TTA TCG CT - #G ACT AAA TTG GAA 6420 Asp Gln Gln Lys Thr Lys Ser Ser Leu Ser Le - #u Thr Lys Leu Glu 135                 1 - #40                 1 - #45 - - CGT TAT CGT TAT GAA ATT TTA TTA GAG GCG GT - #T GCG GCG GAT AGG 6465 Arg Tyr Arg Tyr Glu Ile Leu Leu Glu Ala Va - #l Ala Ala Asp Arg 150                 1 - #55                 1 - #60 - - TTG CCG CTC ATT GAA CTG ACA AAG GAT GAA TT - #T AAA CAT GCT ACG 6510 Leu Pro Leu Ile Glu Leu Thr Lys Asp Glu Ph - #e Lys His Ala Thr 165                 1 - #70                 1 - #75 - - GAA GAA GAT AAA ACC AGA ATT CGC TAT TTG AT - #C ACC GAG CAA TTT 6555 Glu Glu Asp Lys Thr Arg Ile Arg Tyr Leu Il - #e Thr Glu Gln Phe 180                 1 - #85                 1 - #90 - - GAA GCT TGG CAA AAG CAA AAG TAT CAA AAA GA - #A TTA GCT TTG CAA 6600 Glu Ala Trp Gln Lys Gln Lys Tyr Gln Lys Gl - #u Leu Ala Leu Gln 195                 2 - #00                 2 - #05 - - CGT AGA GAA GCA GAA AAA CAA ACG GTT CTA GC - #T AAT ATT CGT AAA 6645 Arg Arg Glu Ala Glu Lys Gln Thr Val Leu Al - #a Asn Ile Arg Lys 210                 2 - #15                 2 - #20 - - TAT GAG GGA ATC AGT CGA GTT GAA AAT GAA AG - #A TTA AAA GAT CTT 6690 Tyr Glu Gly Ile Ser Arg Val Glu Asn Glu Ar - #g Leu Lys Asp Leu 225                 2 - #30                 2 - #35 - - AAA AAA TTA TTT AAT TCG AAA TCG ACT TCT AA - #A CAT GAT GTC TTG 6735 Lys Lys Leu Phe Asn Ser Lys Ser Thr Ser Ly - #s His Asp Val Leu 240                 2 - #45                 2 - #50 - - ACT CAA GAA AAT CGT CAT ATC GAA GCG GTA AA - #T GAG TTG GCG GTG 6780 Thr Gln Glu Asn Arg His Ile Glu Ala Val As - #n Glu Leu Ala Val 255                 2 - #60                 2 - #65 - - TAT AAA TCT CGG TTG AAT GAA GTG GAA AGT GA - #C TTA CGT CAA GCC 6825 Tyr Lys Ser Arg Leu Asn Glu Val Glu Ser As - #p Leu Arg Gln Ala 270                 2 - #75                 2 - #80 - - AAA GAG GAA ATA CAT TTA ATA ACT CAG TTG TT - #T AGA GCC GAT ATT 6870 Lys Glu Glu Ile His Leu Ile Thr Gln Leu Ph - #e Arg Ala Asp Ile 285                 2 - #90                 2 - #95 - - CTG GAG AAG TTG AAA CAA AAT GTT GAA GCG GA - #G AAA CAG CTT TCG 6915 Leu Glu Lys Leu Lys Gln Asn Val Glu Ala Gl - #u Lys Gln Leu Ser 300                 3 - #05                 3 - #10 - - CTC GAA TTA GAA AAA AAT GAG CAG CGT CAA AT - #T GCT TCG GTG ATT 6960 Leu Glu Leu Glu Lys Asn Glu Gln Arg Gln Il - #e Ala Ser Val Ile 315                 3 - #20                 3 - #25 - - CGT GCG CCG GTT TCC GGT ACG GTT CAG CAA CT - #T AAA ACC CAT ACG 7005 Arg Ala Pro Val Ser Gly Thr Val Gln Gln Le - #u Lys Thr His Thr 330                 3 - #35                 3 - #40 - - GTA GGC GGC GTC GTG ACG ACT GCC GAA ACC TT - #G ATG GTA ATT GCT 7050 Val Gly Gly Val Val Thr Thr Ala Glu Thr Le - #u Met Val Ile Ala 345                 3 - #50                 3 - #55 - - CCG GAA GAT GAT GTT TTA GAG GTA ACG GCG TT - #A ATT CAA AAT AAG 7095 Pro Glu Asp Asp Val Leu Glu Val Thr Ala Le - #u Ile Gln Asn Lys 360                 3 - #65                 3 - #70 - - GAT ATC GGT TTT ATC GAG GTC GGT CAG GAT GC - #G GTG ATT AAA GTA 7140 Asp Ile Gly Phe Ile Glu Val Gly Gln Asp Al - #a Val Ile Lys Val 375                 3 - #80                 3 - #85 - - GAA ACT TTT CCT TAT ACT CGT TAC GGC TAT TT - #A ATG GGT AAA GTA 7185 Glu Thr Phe Pro Tyr Thr Arg Tyr Gly Tyr Le - #u Met Gly Lys Val 390                 3 - #95                 4 - #00 - - AAA AAT ATC ACG CTG GAA GCC ATC GAA CAT CC - #G CAA CTC GGT CTA 7230 Lys Asn Ile Thr Leu Glu Ala Ile Glu His Pr - #o Gln Leu Gly Leu 405                 4 - #10                 4 - #15 - - GTT TTT AAC TCG ATT ATT TCT ATT GAT AGA AA - #A ACT TTA TCC GGC 7275 Val Phe Asn Ser Ile Ile Ser Ile Asp Arg Ly - #s Thr Leu Ser Gly 420                 4 - #25                 4 - #30 - - AAA GAC GGC AAA GAA ATT GAA CTT GGA TCA GG - #T ATG AGT GTG ACG 7320 Lys Asp Gly Lys Glu Ile Glu Leu Gly Ser Gl - #y Met Ser Val Thr 435                 4 - #40                 4 - #45 - - GCG GAA ATT AAA ACT GGA GAA CGT AGC GTT AT - #T AGT TAT TTA CTC 7365 Ala Glu Ile Lys Thr Gly Glu Arg Ser Val Il - #e Ser Tyr Leu Leu 450                 4 - #55                 4 - #60 - - AGT CCG TTG GAA GAA TCC GTT TCG GAG AGT TT - #A AGA GAA CGC TAA 7410 Ser Pro Leu Glu Glu Ser Val Ser Glu Ser Le - #u Arg Glu Arg 465                 4 - #70                 4 - #75 - - AGCAGATAAA ACAAGCGGCC ATATTTTCTT ACTTTTTTGC AAAAAACGTA TG - #AAATAT 7470 - - CCGCTTGTCG TTTGTAAAAG ACTATTTATT TACAATAATT TTAGCACCGT TA - #GAAAAT 7530 - - GATCTGACGA GCTTCAAATT GAGCGGAGAG CTGTGCTTGC GGGTTTAGAA AT - #ACGGCT 7590 - - TGCTTCTTGC GGTAAGTCTG AAACCGGTAC GCAAAGGCAA GTTCCGCCGT GG - #TTTGGC 7650 - - TTTAAGTTAT CTTTAAAGGT AACGGGCGCA TCTTGCGTGA GGATAACTTT AT - #CATTGT 7710 - - ACATAGTTTA CCGCCCATTG AACGATACGA ATATTGCGTT TGGTTTTATT TT - #CAATAC 7770 - - TATTTAAAGC TAACCATCGG CTGCCCTTCT TTATTTTTAG CCAATTCATA AC - #CGAAAA 7830 - - CGTAACCCGA TACTGTCATT AAATTGTTTA AGGCGTTTTT CTTTAGCCGA AA - #GAGGTG 7890 - - TTTTTCGTTA CTGATTTATG TTCAACCGTC GGTTGAATTT TATTGCCTTC AG - #CTTGAG 7950 - - TTAAACGCTA AAAAGAATGA TGCTACCGCC GTGCTAAGTA ATTTAATGTG TT - #TCATAA 8010 - - CACCTCGTAA TGAGAGCTAA AAGCCGACTT GATATATTAC GCTATATATT GT - #CAGATT 8070 - - CGGCACAGTT GCAATGACCG CATAACCGTC CGATTCGGCA ATAATCTCGA CT - #TGGCTT 8130 - - CGCCGCAATG AAAATCGCTT CGCCTTGTTG GAGATAAATG GACTCTTCAC CG - #AGGTCG 8190 - - ATAGATACTG CCTTTCATCA CCAATAAGAT ACTTGCACAG TCGGCCGTAA AG - #TTTTCT 8250 - - GTCAAATGCG TTGAATTGCA TATGTTGCAA TGCAAAATCT TTCGCTTCAG GC - #GTCGGA 8310 - - AAGATGAATG AAACCGTCGT TTTCTTGATA AGGCGGAATA ACTTCGGGGT AA - #TCGGGC 8370 - -  - - (2) INFORMATION FOR SEQ ID NO: 2: - -      (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4731 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: - - CTTAACCATT ACAGAACGTT GGTACAAAAA ATTTTACAGG AAAATGATGG AT - #AGTCCTTA     60 - - ACAAAAATTA ATGTTTTATT TCCTATAAAA CATCCGACCA GTATTATTTT TG - #ATTAAAA     120 - - AAGAACAAAC AGATCATGAC AAACGTTTGC CTTGTTTTCC TTCACAAAAA TA - #TTATGGT     180 - - TTTTATTTAG AATAAATTAT CTATATTCAT TTTTTAGGGA ATGGGAGGGA TG - # 232 - - ATG CTA AAA AAT GAT TTT AAC GTA TTG GGA CA - #A ATT GCT TGG TTA 27 - #7 Met Leu Lys Asn Asp Phe Asn Val Leu Gly Gl - #n Ile Ala Trp Leu 1               5  - #                 10 - #                 15 - - TGG GCA AAT TCT CCA ATG CAC CGA AAT TGG TC - #A GTT TCA CTG TTA 32 - #2 Trp Ala Asn Ser Pro Met His Arg Asn Trp Se - #r Val Ser Leu Leu 20 - #                 25 - #                 30 - - ATG AAG AAT GTT ATT CCT GCA ATT GAA AAT GA - #C CAA TAT TTG TTA 36 - #7 Met Lys Asn Val Ile Pro Ala Ile Glu Asn As - #p Gln Tyr Leu Leu 35 - #                 40 - #                 45 - - CTA GTT GAT GAT GGT TTT CCT ATT GCA TAT TG - #C AGT TGG GCC AAA 41 - #2 Leu Val Asp Asp Gly Phe Pro Ile Ala Tyr Cy - #s Ser Trp Ala Lys 50 - #                 55 - #                 60 - - TTA ACT CTA GAG AGT GAG GCT CGC TAT GTA AA - #G GAC ACC AAT TCA 45 - #7 Leu Thr Leu Glu Ser Glu Ala Arg Tyr Val Ly - #s Asp Thr Asn Ser 65 - #                 70 - #                 75 - - TTA AAA ATA GAT GAT TGG AAT GCA GGA GAT CG - #T ATA TGG ATC ATT 50 - #2 Leu Lys Ile Asp Asp Trp Asn Ala Gly Asp Ar - #g Ile Trp Ile Ile 80 - #                 85 - #                 90 - - GAT TGG ATT GCC CCA TTC GGG GAT TCA TCT CT - #A TTG TAT AAA CAT 54 - #7 Asp Trp Ile Ala Pro Phe Gly Asp Ser Ser Le - #u Leu Tyr Lys His 95 - #                100 - #                105 - - ATG AGA CAA CGT TTT CCA TAC GAT ATT GGA AG - #G GCA ATT AGA ATC 59 - #2 Met Arg Gln Arg Phe Pro Tyr Asp Ile Gly Ar - #g Ala Ile Arg Ile 110  - #               115  - #               120 - - TAT CCT AGC AAA AAA GAT ACT GGA AAA ATC AT - #A TAT TTA AAA GGA 63 - #7 Tyr Pro Ser Lys Lys Asp Thr Gly Lys Ile Il - #e Tyr Leu Lys Gly 125  - #               130  - #               135 - - GGA AAA ATA ACA AAA AAA GTA GCT GAA AAG AC - #A TTT CTT CAG TAT 68 - #2 Gly Lys Ile Thr Lys Lys Val Ala Glu Lys Th - #r Phe Leu Gln Tyr 140  - #               145  - #               150 - - GAG CAA GAG TTA ATA ACA GCT CTA CAA TAATATCTT - #T AAATGATCAA 729 Glu Gln Glu Leu Ile Thr Ala Leu Gln 155 - - TTATATAAAG GAGACTCTTTT      ATG TCA AAA AT - #C ACT TTG TCA TCA 77 - #4 - #          Met Ser Lys Ile Thr - # Leu Ser Ser - #           1       - #        5 - - TTA AAA TCG TCC TTA CAA CAA GGA TTG AAA AA - #T GGG AAA AAC AAG 81 - #9 Leu Lys Ser Ser Leu Gln Gln Gly Leu Lys As - #n Gly Lys Asn Lys 10             - #     15             - #     20 - - TTA AAT CAA GCA GGT ACA ACA CTG AAG AAT GG - #T TTA ACT CAA ACT 86 - #4 Leu Asn Gln Ala Gly Thr Thr Leu Lys Asn Gl - #y Leu Thr Gln Thr 25             - #     30             - #     35 - - GGT CAT TCT CTA CAG AAT GGG GCT AAA AAA TT - #A ATC TTA TAT ATT 90 - #9 Gly His Ser Leu Gln Asn Gly Ala Lys Lys Le - #u Ile Leu Tyr Ile 40             - #     45             - #     50 - - CCT CAA GGC TAT GAT TCG GGT CAA GGA AAT GG - #A GTT CAA GAT TTA 95 - #4 Pro Gln Gly Tyr Asp Ser Gly Gln Gly Asn Gl - #y Val Gln Asp Leu 55             - #     60             - #     65 - - GTT AAA GCT GCT AAT GAT TTA GGT ATT GAA GT - #A TGG CGA GAA GAA 99 - #9 Val Lys Ala Ala Asn Asp Leu Gly Ile Glu Va - #l Trp Arg Glu Glu 70             - #     75             - #     80 - - CGC AGC AAT TTG GAC ATT GCA AAA ACT AGC TT - #T GAT ACA ACT CAG 1044 Arg Ser Asn Leu Asp Ile Ala Lys Thr Ser Ph - #e Asp Thr Thr Gln 85             - #     90             - #     95 - - AAA ATT CTA GGT TTT ACT GAT AGA GGA ATT GT - #A TTA TTT GCA CCT 1089 Lys Ile Leu Gly Phe Thr Asp Arg Gly Ile Va - #l Leu Phe Ala Pro 100              - #   105              - #   110 - - CAG CTA GAT AAT TTA TTA AAG AAG AAT CCT AA - #A ATT GGC AAT ACA 1134 Gln Leu Asp Asn Leu Leu Lys Lys Asn Pro Ly - #s Ile Gly Asn Thr 115              - #   120              - #   125 - - TTA GGA AGT GCT TCT AGC ATC TCA CAA AAT AT - #A GGT AAA GCC AAT 1179 Leu Gly Ser Ala Ser Ser Ile Ser Gln Asn Il - #e Gly Lys Ala Asn 130              - #   135              - #   140 - - ACT GTA TTA GGT GGT ATT CAA TCT ATT TTA GG - #A TCT GTT TTA TCT 1224 Thr Val Leu Gly Gly Ile Gln Ser Ile Leu Gl - #y Ser Val Leu Ser 145              - #   150              - #   155 - - GGA GTA AAT CTG AAT GAA TTA CTT CAA AAT AA - #A GAT CCT AAT CAA 1269 Gly Val Asn Leu Asn Glu Leu Leu Gln Asn Ly - #s Asp Pro Asn Gln 160              - #   165              - #   170 - - TTA GAA CTT GCA AAA GCA GGG CTA GAA CTG AC - #T AAT GAA TTA GTT 1314 Leu Glu Leu Ala Lys Ala Gly Leu Glu Leu Th - #r Asn Glu Leu Val 175              - #   180              - #   185 - - GGT AAT ATT GCT AGC TCG GTG CAA ACT GTA GA - #T GCA TTT GCA GAA 1359 Gly Asn Ile Ala Ser Ser Val Gln Thr Val As - #p Ala Phe Ala Glu 190              - #   195              - #   200 - - CAA ATA TCT AAA CTA GGT TCA CAT TTA CAG AA - #T GTG AAA GGA TTA 1404 Gln Ile Ser Lys Leu Gly Ser His Leu Gln As - #n Val Lys Gly Leu 205              - #   210              - #   215 - - GGA GGA TTG AGT AAT AAA TTA CAA AAT CTA CC - #A GAT CTA GGA AAA 1449 Gly Gly Leu Ser Asn Lys Leu Gln Asn Leu Pr - #o Asp Leu Gly Lys 220              - #   225              - #   230 - - GCA AGT TTA GGT TTG GAC ATT ATC TCT GGT TT - #A CTT TCT GGA GCA 1494 Ala Ser Leu Gly Leu Asp Ile Ile Ser Gly Le - #u Leu Ser Gly Ala 235              - #   240              - #   245 - - TCT GCA GGT CTC ATT TTA GCA GAT AAA GAG GC - #T TCA ACA GAA AAG 1539 Ser Ala Gly Leu Ile Leu Ala Asp Lys Glu Al - #a Ser Thr Glu Lys 250              - #   255              - #   260 - - AAA GCT GCC GCA GGT GTA GAA TTT GCT AAC CA - #A ATT ATA GGT AAT 1584 Lys Ala Ala Ala Gly Val Glu Phe Ala Asn Gl - #n Ile Ile Gly Asn 265              - #   270              - #   275 - - GTA ACA AAA GCG GTC TCA TCT TAC ATT CTT GC - #C CAA CGA GTC GCT 1629 Val Thr Lys Ala Val Ser Ser Tyr Ile Leu Al - #a Gln Arg Val Ala 280              - #   285              - #   290 - - TCA GGT TTG TCT TCA ACT GGT CCT GTC GCT GC - #A TTA ATC GCA TCT 1674 Ser Gly Leu Ser Ser Thr Gly Pro Val Ala Al - #a Leu Ile Ala Ser 295              - #   300              - #   305 - - ACA GTT GCA CTA GCT GTT AGC CCT CTT TCA TT - #C TTA AAT GTA GCT 1719 Thr Val Ala Leu Ala Val Ser Pro Leu Ser Ph - #e Leu Asn Val Ala 310              - #   315              - #   320 - - GAT AAG TTT AAA CAA GCT GAT TTA ATC AAA TC - #A TAT TCT GAA CGC 1764 Asp Lys Phe Lys Gln Ala Asp Leu Ile Lys Se - #r Tyr Ser Glu Arg 325              - #   330              - #   335 - - TTC CAA AAA TTA GGA TAT GAT GGA GAT CGT TT - #A TTA GCT GAT TTT 1809 Phe Gln Lys Leu Gly Tyr Asp Gly Asp Arg Le - #u Leu Ala Asp Phe 340              - #   345              - #   350 - - CAC CGT GAG ACA GGA ACT ATT GAT GCT TCT GT - #A ACA ACA ATT AAC 1854 His Arg Glu Thr Gly Thr Ile Asp Ala Ser Va - #l Thr Thr Ile Asn 355              - #   360              - #   365 - - ACT GCT TTA GCA GCT ATC TCC GGT GGA GTT GG - #A GCT GCA AGC GCG 1899 Thr Ala Leu Ala Ala Ile Ser Gly Gly Val Gl - #y Ala Ala Ser Ala 370              - #   375              - #   380 - - GGT TCT CTA GTC GGA GCT CCA GTT GCG TTA CT - #C GTT GCT GGT GTT 1944 Gly Ser Leu Val Gly Ala Pro Val Ala Leu Le - #u Val Ala Gly Val 385              - #   390              - #   395 - - ACG GGA CTT ATT ACA ACT ATT CTA GAA TAT TC - #T AAA CAA GCC ATG 1989 Thr Gly Leu Ile Thr Thr Ile Leu Glu Tyr Se - #r Lys Gln Ala Met 400              - #   405              - #   410 - - TTT GAA CAT GTT GCA AAT AAG GTT CAT GAC AG - #A ATA GTT GAA TGG 2034 Phe Glu His Val Ala Asn Lys Val His Asp Ar - #g Ile Val Glu Trp 415              - #   420              - #   425 - - GAG AAA AAA CAT AAT AAA AAC TAT TTT GAG CA - #A GGT TAT GAT TCT 2079 Glu Lys Lys His Asn Lys Asn Tyr Phe Glu Gl - #n Gly Tyr Asp Ser 430              - #   435              - #   440 - - CGT CAT TTA GCT GAT TTA CAA GAC AAT ATG AA - #G TTT CTT ATC AAT 2124 Arg His Leu Ala Asp Leu Gln Asp Asn Met Ly - #s Phe Leu Ile Asn 445              - #   450              - #   455 - - TTA AAT AAA GAA CTT CAG GCT GAA CGC GTA GT - #A GCT ATT ACC CAA 2169 Leu Asn Lys Glu Leu Gln Ala Glu Arg Val Va - #l Ala Ile Thr Gln 460              - #   465              - #   470 - - CAA AGA TGG GAT AAC CAA ATT GGA GAC CTA GC - #G GCA ATT AGC CGT 2214 Gln Arg Trp Asp Asn Gln Ile Gly Asp Leu Al - #a Ala Ile Ser Arg 475              - #   480              - #   485 - - AGA ACG GAT AAA ATT TCC AGT GGA AAA GCT TA - #T GTG GAT GCT TTT 2259 Arg Thr Asp Lys Ile Ser Ser Gly Lys Ala Ty - #r Val Asp Ala Phe 490              - #   495              - #   500 - - GAG GAG GGG CAA CAC CAG TCC TAC GAT TCA TC - #C GTA CAG CTA GAT 2304 Glu Glu Gly Gln His Gln Ser Tyr Asp Ser Se - #r Val Gln Leu Asp 505              - #   510              - #   515 - - AAC AAA AAC GGT ATT ATT AAT ATT AGT AAT AC - #A AAT AGA AAG ACA 2349 Asn Lys Asn Gly Ile Ile Asn Ile Ser Asn Th - #r Asn Arg Lys Thr 520              - #   525              - #   530 - - CAA AGT GTT TTA TTC AGA ACT CCA TTA CTA AC - #T CCA GGT GAA GAG 2394 Gln Ser Val Leu Phe Arg Thr Pro Leu Leu Th - #r Pro Gly Glu Glu 535              - #   540              - #   545 - - AAT CGG GAA CGT ATT CAG GAA GGT AAA AAT TC - #T TAT ATT ACA AAA 2439 Asn Arg Glu Arg Ile Gln Glu Gly Lys Asn Se - #r Tyr Ile Thr Lys 550              - #   555              - #   560 - - TTA CAT ATA CAA AGA GTT GAC AGT TGG ACT GT - #A ACA GAT GGT GAT 2484 Leu His Ile Gln Arg Val Asp Ser Trp Thr Va - #l Thr Asp Gly Asp 565              - #   570              - #   575 - - GCT AGC TCA AGC GTA GAT TTC ACT AAT GTA GT - #A CAA CGA ATC GCT 2529 Ala Ser Ser Ser Val Asp Phe Thr Asn Val Va - #l Gln Arg Ile Ala 580              - #   585              - #   590 - - GTG AAA TTT GAT GAT GCA GGT AAC ATT ATA GA - #A TCT AAA GAT ACT 2574 Val Lys Phe Asp Asp Ala Gly Asn Ile Ile Gl - #u Ser Lys Asp Thr 595              - #   600              - #   605 - - AAA ATT ATC GCA AAT TTA GGT GCT GGT AAC GA - #T AAT GTA TTT GTT 2619 Lys Ile Ile Ala Asn Leu Gly Ala Gly Asn As - #p Asn Val Phe Val 610              - #   615              - #   620 - - GGG TCA AGT ACT ACC GTT ATT GAT GGC GGG GA - #C GGA CAT GAT CGA 2664 Gly Ser Ser Thr Thr Val Ile Asp Gly Gly As - #p Gly His Asp Arg 625              - #   630              - #   635 - - GTT CAC TAC AGT AGA GGA GAA TAT GGC GCA TT - #A GTT ATT GAT GCT 2709 Val His Tyr Ser Arg Gly Glu Tyr Gly Ala Le - #u Val Ile Asp Ala 640              - #   645              - #   650 - - ACA GCC GAG ACA GAA AAA GGC TCA TAT TCA GT - #A AAA CGC TAT GTC 2754 Thr Ala Glu Thr Glu Lys Gly Ser Tyr Ser Va - #l Lys Arg Tyr Val 655              - #   660              - #   665 - - GGA GAC AGT AAA GCA TTA CAT GAA ACA ATT GC - #C ACC CAC CAA ACA 2799 Gly Asp Ser Lys Ala Leu His Glu Thr Ile Al - #a Thr His Gln Thr 670              - #   675              - #   680 - - AAT GTT GGT AAT CGT GAA GAA AAA ATT GAA TA - #T CGT CGT GAA GAT 2844 Asn Val Gly Asn Arg Glu Glu Lys Ile Glu Ty - #r Arg Arg Glu Asp 685              - #   690              - #   695 - - GAT CGT TTT CAT ACT GGT TAT ACT GTG ACG GA - #C TCA CTC AAA TCA 2889 Asp Arg Phe His Thr Gly Tyr Thr Val Thr As - #p Ser Leu Lys Ser 700              - #   705              - #   710 - - GTT GAA GAG ATC ATT GGT TCA CAA TTT AAT GA - #T ATT TTC AAA GGA 2934 Val Glu Glu Ile Ile Gly Ser Gln Phe Asn As - #p Ile Phe Lys Gly 715              - #   720              - #   725 - - AGC CAA TTT GAT GAT GTG TTC CAT GGT GGT AA - #T GGT GTA GAC ACT 2979 Ser Gln Phe Asp Asp Val Phe His Gly Gly As - #n Gly Val Asp Thr 730              - #   735              - #   740 - - ATT GAT GGT AAC GAT GGT GAC GAT CAT TTA TT - #T GGT GGC GCA GGC 3024 Ile Asp Gly Asn Asp Gly Asp Asp His Leu Ph - #e Gly Gly Ala Gly 745              - #   750              - #   755 - - GAT GAT GTT ATC GAT GGA GGA AAC GGT AAC AA - #T TTC CTT GTT GGA 3069 Asp Asp Val Ile Asp Gly Gly Asn Gly Asn As - #n Phe Leu Val Gly 760              - #   765              - #   770 - - GGA ACC GGT AAT GAT ATT ATC TCG GGA GGT AA - #A GAT AAT GAT ATT 3114 Gly Thr Gly Asn Asp Ile Ile Ser Gly Gly Ly - #s Asp Asn Asp Ile 775              - #   780              - #   785 - - TAT GTC CAT AAA ACA GGC GAT GGA AAT GAT TC - #T ATT ACA GAC TCT 3159 Tyr Val His Lys Thr Gly Asp Gly Asn Asp Se - #r Ile Thr Asp Ser 790              - #   795              - #   800 - - GGC GGA CAA GAT AAA CTG GCA TTT TCG GAT GT - #A AAT CTT AAA GAC 3204 Gly Gly Gln Asp Lys Leu Ala Phe Ser Asp Va - #l Asn Leu Lys Asp 805              - #   810              - #   815 - - CTC ACC TTT AAG AAA GTA GAT TCT TCT CTC GA - #A ATC ATT AAT CAA 3249 Leu Thr Phe Lys Lys Val Asp Ser Ser Leu Gl - #u Ile Ile Asn Gln 820              - #   825              - #   830 - - AAA GGA GAA AAA GTT CGT ATT GGG AAT TGG TT - #C TTA GAA GAT GAT 3294 Lys Gly Glu Lys Val Arg Ile Gly Asn Trp Ph - #e Leu Glu Asp Asp 835              - #   840              - #   845 - - TTG GCT AGC ACA GTT GCT AAC TAT AAA GCT AC - #G AAT GAC CGA AAA 3339 Leu Ala Ser Thr Val Ala Asn Tyr Lys Ala Th - #r Asn Asp Arg Lys 850              - #   855              - #   860 - - ATT GAG GAA ATT ATT GGT AAA GGA GGA GAA CG - #T ATT ACA TCA GAA 3384 Ile Glu Glu Ile Ile Gly Lys Gly Gly Glu Ar - #g Ile Thr Ser Glu 865              - #   870              - #   875 - - CAA GTT GAT AAA CTG ATT AAG GAG GGT AAC AA - #T CAA ATC TCT GCA 3429 Gln Val Asp Lys Leu Ile Lys Glu Gly Asn As - #n Gln Ile Ser Ala 880              - #   885              - #   890 - - GAA GCA TTA TCC AAA GTT GTG AAT GAT TAC AA - #T ACG AGT AAA GAT 3474 Glu Ala Leu Ser Lys Val Val Asn Asp Tyr As - #n Thr Ser Lys Asp 895              - #   900              - #   905 - - AGA CAG AAC GTA TCT AAT AGC TTA GCA AAA TT - #G ATT TCT TCA GTC 3519 Arg Gln Asn Val Ser Asn Ser Leu Ala Lys Le - #u Ile Ser Ser Val 910              - #   915              - #   920 - - GGG AGC TTT ACG TCT TCC TCA GAC TTT AGG AA - #T AAT TTA GGA ACA 3564 Gly Ser Phe Thr Ser Ser Ser Asp Phe Arg As - #n Asn Leu Gly Thr 925              - #   930              - #   935 - - TAT GTT CCT TCA TCA ATA GAT GTC TCG AAT AA - #T ATT CAA TTA GCT 3609 Tyr Val Pro Ser Ser Ile Asp Val Ser Asn As - #n Ile Gln Leu Ala 940              - #   945              - #   950 - - AGA GCC GCT TAA            - #                  - # - #     3621 Arg Ala Ala 955 - - TATTCAAATC ATAGCAATCC TATGGTGTAA ATTATAGGAT TGTTATTTTT TT - #AAAGGA 3681 - - AGTTATGGAA CCCAATAAAA ATAAGGATCT TGGTTTAGCT GTAGAAAATC AA - #ACCTAA 3741 - - TGACAGTTCC CGTTTAAAAT TACCGTGTCT GTCAGATTAA TTTGAGCTTA AA - #TTCTTT 3801 - - TGCCCAAATC CGTTTTCCAT CAAGTAATGT TGCCATCGGT GTTCTGCCAC AG - #CACACT 3861 - - TCCTTGATGT GTTCGATGGT GATTATAATA CATTCATCTA AATCAGCTTG TA - #ATGTCG 3921 - - AAATCCGTAT ATATTTTCTT CCTAAATGCG ACTTGGTAAA ATTCTTGTAA GA - #TAGTCT 3981 - - TGAAAACGTT CACAGATACC ATTCGTCTGT GGATGCTTCA CTTTCGTTTT AG - #TATGCT 4041 - - ATGTCATTTA TCGCTAAATA AAGCTCATAA TCGTGATTTT CCACTTTGCC AC - #AATATT 4101 - - CTGCCACGGT CGGTGAGAAT ACGCAACATC GGTAATCCTT GGGCTTCAAA GA - #ACGGCA 4161 - - ACTTTATGAT TGAGCATATC TGCAGCGGCA ATTGCGGTTT TCATTGTGTA GA - #GCTTTG 4221 - - AAAGCAACCT TACTATAAGT ATCAACAAAT GTTTGCTGAT AAATGCGTCC AA - #CACCTT 4281 - - AAATTACCTA CATAAAAGGT ATCTTGTGAA CCTAAATAGC CCGGATGAGC GG - #TTTCAA 4341 - - TCTCCACTCG ATATATCATC CTCTTTCTTA CGTTCTAGGG CTTGGACTTG AC - #TTTCAT 4401 - - AGAATAATGC CTTTCTCAGC CACTTCTTTC TCTAGTGCAT TTAAACGCTG TT - #TAAAGT 4461 - - GTAAGATTAT GACGTAGCCA AATGGAACGA ACACCACCGG CTGAAACAAA CA - #CACCTT 4521 - - TTGCGAAGTT CGTTACTCAC TCGAACTTGT CCGTAAGCTG GAAAATCTAG AG - #CAAATT 4581 - - ACAACAGCTT GCTCAATGTG CTCGTCTACT CGATTTTTGA TATTCGGTAC CC - #GACGAG 4641 - - TGCTTAAGTA ATGCTTCAAC ACCGCCTTGC GCTACGGCTT GTTGATAGCG AT - #AGAATG 4701 - - TCTCGGCTCA TTCCCATCGC TTTACAAGCT         - #                  - # 4731 - -  - - (2) INFORMATION FOR SEQ ID NO: 3: - -      (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4190 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3: - - GTAGATATTC TTTTAATATC AAACAACTAT TGTTATTTGT CTGAGTGTAG AT - #ATGTAGCA     60 - - TTGTGTATTT CTTTATTTAC AACTCTAATC TTAATCTAAA AAGATTTCTA TA - #TTTTCTT     120 - - GTAAGAAATT TTGTTAAAAT CCGACTAACT ATATAATTAA CGGTTCTTAA AG - #TGGATAA     180 - - TAATAAAATT ATGAGTTATA AAAATGTTAA AAATTTAACA GATGATTTTA CA - #ACTTTAG     240 - - GCATATCGCT TGGTTGTGGG CTAATTCTCC GTTACATAAG GAGTGGTCTA TC - #TCTTTGT     300 - - TACTAAGAAT ATTTTGCCAG CCATTCAACA TGATCAATAT ATTTTACTTA TG - #CGAGATG     360 - - GTTCCCTGTA GCGTTTTGTA GTTGGGCAAA TTTAACGTTA ACTAATGAAG TG - #AAGTATG     420 - - ACGTGATGTG ACGTCATTGA CTTTTGAAGA TTGGAATTCA GGAGAACGAA AA - #TGGTTGA     480 - - CGACTGGATT GCGCCATTTG GGGATAACAA TACGCTTTAT AGATATATGC GT - #AAAAAAT     540 - - TCCTAATGAA GTATTCCGGG CCATTCGAGT ATATCCTGGT TCTACAGAAG CG - #AAAATCA     600 - - TCATGTTCAA GGAGGACAAA TTAATAAATT TACAGCTAAA AAATTAATAC AA - #CAATATC     660 - - GGAAGAACTT ATTCAAGTTC TTAACAATCA CAAAAAAATT GTAAGAGGAT AA - #AAT 715 - - ATG AGT ACT TGG TCA AGC ATG TTA GCC GAC TT - #A AAA AAA CGG GCT 76 - #0 Met Ser Thr Trp Ser Ser Met Leu Ala Asp Le - #u Lys Lys Arg Ala 1               5  - #                 10 - #                 15 - - GAA GAA GCC AAA AGA CAA GCC AAA AAA GGC TA - #C GAT GTA ACT AAA 80 - #5 Glu Glu Ala Lys Arg Gln Ala Lys Lys Gly Ty - #r Asp Val Thr Lys 20 - #                 25 - #                 30 - - AAT GGT TTG CAA TAT GGG GTG AGT CAA GCA AA - #A TTA CAA GCA TTA 85 - #0 Asn Gly Leu Gln Tyr Gly Val Ser Gln Ala Ly - #s Leu Gln Ala Leu 35 - #                 40 - #                 45 - - GCA GCT GGT AAA GCC GTT CAA AAG TAC GGT AA - #T AAA TTA GTT TTA 89 - #5 Ala Ala Gly Lys Ala Val Gln Lys Tyr Gly As - #n Lys Leu Val Leu 50 - #                 55 - #                 60 - - GTT ATT CCA AAA GAG TAT GAC GGA AGT GTT GG - #T AAC GGT TTC TTT 94 - #0 Val Ile Pro Lys Glu Tyr Asp Gly Ser Val Gl - #y Asn Gly Phe Phe 65 - #                 70 - #                 75 - - GAT TTA GTA AAA GCA GCT GAG GAA TTA GGC AT - #T CAA GTT AAA TAT 98 - #5 Asp Leu Val Lys Ala Ala Glu Glu Leu Gly Il - #e Gln Val Lys Tyr 80 - #                 85 - #                 90 - - GTT AAC CGT AAT GAA TTG GAA GTT GCC CAT AA - #A AGT TTA GGT ACC 1030 Val Asn Arg Asn Glu Leu Glu Val Ala His Ly - #s Ser Leu Gly Thr 95 - #                100 - #                105 - - GCA GAC CAA TTC TTG GGT TTA ACA GAA CGT GG - #A CTT ACT TTA TTT 1075 Ala Asp Gln Phe Leu Gly Leu Thr Glu Arg Gl - #y Leu Thr Leu Phe 110  - #               115  - #               120 - - GCA CCG CAA CTA GAT CAG TTC TTA CAA AAA CA - #T TCA AAA ATT TCT 1120 Ala Pro Gln Leu Asp Gln Phe Leu Gln Lys Hi - #s Ser Lys Ile Ser 125  - #               130  - #               135 - - AAC GTA GTG GGC AGT TCT ACT GGT GAT GCA GT - #A AGT AAA CTT GCT 1165 Asn Val Val Gly Ser Ser Thr Gly Asp Ala Va - #l Ser Lys Leu Ala 140  - #               145  - #               150 - - AAG AGT CAA ACT ATT ATT TCA GGA ATT CAA TC - #T GTA TTA GGT ACT 1210 Lys Ser Gln Thr Ile Ile Ser Gly Ile Gln Se - #r Val Leu Gly Thr 155  - #               160  - #               165 - - GTA TTA GCA GGT ATT AAT CTT AAT GAA GCT AT - #T ATT AGT GGC GGT 1255 Val Leu Ala Gly Ile Asn Leu Asn Glu Ala Il - #e Ile Ser Gly Gly 170  - #               175  - #               180 - - TCA GAG CTC GAA TTA GCT GAA GCT GGT GTT TC - #T TTA GCC TCT GAG 1300 Ser Glu Leu Glu Leu Ala Glu Ala Gly Val Se - #r Leu Ala Ser Glu 185  - #               190  - #               195 - - CTG CTT AGT AAT ATT GCT AAA GGT ACA ACA AC - #A ATA GAT GCT TTC 1345 Leu Leu Ser Asn Ile Ala Lys Gly Thr Thr Th - #r Ile Asp Ala Phe 200  - #               205  - #               210 - - ACT ACA CAA ATC CAG AAC TTT GGG AAA TTA GT - #G GAA AAT GCT AAA 1390 Thr Thr Gln Ile Gln Asn Phe Gly Lys Leu Va - #l Glu Asn Ala Lys 215  - #               220  - #               225 - - GGG TTA GGT GGT GTT GGC CGC CAA TTA CAG AA - #T ATT TCA GGT TCT 1435 Gly Leu Gly Gly Val Gly Arg Gln Leu Gln As - #n Ile Ser Gly Ser 230  - #               235  - #               240 - - GCA TTA AGC AAA ACT GGA TTA GGT TTG GAT AT - #T ATC TCA AGC TTA 1480 Ala Leu Ser Lys Thr Gly Leu Gly Leu Asp Il - #e Ile Ser Ser Leu 245  - #               250  - #               255 - - CTT TCA GGA GTA ACT GCA AGT TTT GCT TTA GC - #G AAT AAG AAT GCT 1525 Leu Ser Gly Val Thr Ala Ser Phe Ala Leu Al - #a Asn Lys Asn Ala 260  - #               265  - #               270 - - TCA ACA AGC ACT AAA GTT GCT GCT GGC TTT GA - #A CTC TCA AAT CAA 1570 Ser Thr Ser Thr Lys Val Ala Ala Gly Phe Gl - #u Leu Ser Asn Gln 275  - #               280  - #               285 - - GTA ATT GGT GGT ATT ACG AAA GCA GTA TCA AG - #C TAT ATT CTT GCA 1615 Val Ile Gly Gly Ile Thr Lys Ala Val Ser Se - #r Tyr Ile Leu Ala 290  - #               295  - #               300 - - CAG CGT TTA GCT GCT GGT TTA TCT TCG ACA GG - #T CCT GCT GCA GCA 1660 Gln Arg Leu Ala Ala Gly Leu Ser Ser Thr Gl - #y Pro Ala Ala Ala 305  - #               310  - #               315 - - CTA ATT GCG TCT AGT ATT TCT TTA GCA ATC AG - #T CCA TTG GCG TTT 1705 Leu Ile Ala Ser Ser Ile Ser Leu Ala Ile Se - #r Pro Leu Ala Phe 320  - #               325  - #               330 - - TTA CGT GTA GCT GAT AAT TTT AAT CGT TCT AA - #A GAA ATT GGC GAA 1750 Leu Arg Val Ala Asp Asn Phe Asn Arg Ser Ly - #s Glu Ile Gly Glu 335  - #               340  - #               345 - - TTT GCT GAA CGT TTC AAA AAA TTG GGC TAT GA - #C GGC GAT AAA CTA 1795 Phe Ala Glu Arg Phe Lys Lys Leu Gly Tyr As - #p Gly Asp Lys Leu 350  - #               355  - #               360 - - CTT TCA GAG TTT TAT CAC GAA GCT GGT ACT AT - #T GAT GCC TCA ATT 1840 Leu Ser Glu Phe Tyr His Glu Ala Gly Thr Il - #e Asp Ala Ser Ile 365  - #               370  - #               375 - - ACT ACA ATT AGT ACA GCA CTT TCT GCT ATC GC - #A GCT GGA ACG GCC 1885 Thr Thr Ile Ser Thr Ala Leu Ser Ala Ile Al - #a Ala Gly Thr Ala 380  - #               385  - #               390 - - GCC GCG AGT GCA GGT GCA TTA GTT GGC GCA CC - #A ATT ACT TTG TTG 1930 Ala Ala Ser Ala Gly Ala Leu Val Gly Ala Pr - #o Ile Thr Leu Leu 395  - #               400  - #               405 - - GTT ACT GGT ATC ACA GGA TTA ATT TCT GGT AT - #T TTA GAG TTC TCT 1975 Val Thr Gly Ile Thr Gly Leu Ile Ser Gly Il - #e Leu Glu Phe Ser 410  - #               415  - #               420 - - AAA CAA CCA ATG TTA GAT CAT GTT GCA TCG AA - #A ATT GGT AAC AAA 2020 Lys Gln Pro Met Leu Asp His Val Ala Ser Ly - #s Ile Gly Asn Lys 425  - #               430  - #               435 - - ATT GAC GAA TGG GAG AAA AAA TAC GGT AAA AA - #T TAC TTC GAG AAT 2065 Ile Asp Glu Trp Glu Lys Lys Tyr Gly Lys As - #n Tyr Phe Glu Asn 440  - #               445  - #               450 - - GGC TAT GAT GCT CGT CAT AAA GCT TTC TTA GA - #A GAT TCA TTC TCA 2110 Gly Tyr Asp Ala Arg His Lys Ala Phe Leu Gl - #u Asp Ser Phe Ser 455  - #               460  - #               465 - - TTA TTG TCT AGT TTT AAT AAA CAA TAT GAA AC - #T GAA AGA GCT GTT 2155 Leu Leu Ser Ser Phe Asn Lys Gln Tyr Glu Th - #r Glu Arg Ala Val 470  - #               475  - #               480 - - TTA ATT ACA CAA CAA CGT TGG GAT GAA TAT AT - #T GGC GAA CTT GCG 2200 Leu Ile Thr Gln Gln Arg Trp Asp Glu Tyr Il - #e Gly Glu Leu Ala 485  - #               490  - #               495 - - GGT ATT ACT GGC AAA GGT GAC AAA CTC TCT AG - #T GGT AAG GCG TAT 2245 Gly Ile Thr Gly Lys Gly Asp Lys Leu Ser Se - #r Gly Lys Ala Tyr 500  - #               505  - #               510 - - GTA GAT TAC TTT CAA GAA GGT AAA TTA TTA GA - #G AAA AAA CCT GAT 2290 Val Asp Tyr Phe Gln Glu Gly Lys Leu Leu Gl - #u Lys Lys Pro Asp 515  - #               520  - #               525 - - GAC TTT AGC AAA GTA GTT TTC GAT CCA ACT AA - #G GGC GAA ATT GAT 2335 Asp Phe Ser Lys Val Val Phe Asp Pro Thr Ly - #s Gly Glu Ile Asp 530  - #               535  - #               540 - - ATT TCA AAT AGC CAA ACG TCA ACG TTG TTA AA - #A TTT GTT ACG CCA 2380 Ile Ser Asn Ser Gln Thr Ser Thr Leu Leu Ly - #s Phe Val Thr Pro 545  - #               550  - #               555 - - TTA TTA ACA CCA GGT ACA GAG TCA CGT GAA AG - #A ACT CAA ACA GGT 2425 Leu Leu Thr Pro Gly Thr Glu Ser Arg Glu Ar - #g Thr Gln Thr Gly 560  - #               565  - #               570 - - AAA TAT GAA TAT ATC ACG AAG TTA GTT GTA AA - #A GGT AAA GAT AAA 2470 Lys Tyr Glu Tyr Ile Thr Lys Leu Val Val Ly - #s Gly Lys Asp Lys 575  - #               580  - #               585 - - TGG GTT GTT AAT GGC GTT AAA GAT AAA GGT GC - #C GTT TAT GAT TAT 2515 Trp Val Val Asn Gly Val Lys Asp Lys Gly Al - #a Val Tyr Asp Tyr 590  - #               595  - #               600 - - ACT AAT TTA ATT CAA CAT GCT CAT ATT AGT TC - #A TCA GTA GCA CGT 2560 Thr Asn Leu Ile Gln His Ala His Ile Ser Se - #r Ser Val Ala Arg 605  - #               610  - #               615 - - GGT GAA GAA TAC CGT GAA GTT CGT TTG GTA TC - #T CAT CTA GGC AAT 2605 Gly Glu Glu Tyr Arg Glu Val Arg Leu Val Se - #r His Leu Gly Asn 620  - #               625  - #               630 - - GGT AAT GAC AAA GTG TTC TTA GTC GCG GGT TC - #C GCA GAA ATT CAC 2650 Gly Asn Asp Lys Val Phe Leu Val Ala Gly Se - #r Ala Glu Ile His 635  - #               640  - #               645 - - GCT GGT GAA GGT CAT GAT GTG GTT TAT TAT GA - #T AAA ACC GAT ACA 2695 Ala Gly Glu Gly His Asp Val Val Tyr Tyr As - #p Lys Thr Asp Thr 650  - #               655  - #               660 - - GGT CTT TTA GTA ATT GAT GGA ACC AAA GCG AC - #T GAA CAA GGG CGT 2740 Gly Leu Leu Val Ile Asp Gly Thr Lys Ala Th - #r Glu Gln Gly Arg 665  - #               670  - #               675 - - TAT TCT GTT ACG CGC GAA TTG AGT GGT GCT AC - #A AAA ATC CTG AGA 2785 Tyr Ser Val Thr Arg Glu Leu Ser Gly Ala Th - #r Lys Ile Leu Arg 680  - #               685  - #               690 - - GAA GTA ATA AAA AAT CAA AAA TCT GCT GTT GG - #T AAA CGT GAA GAA 2830 Glu Val Ile Lys Asn Gln Lys Ser Ala Val Gl - #y Lys Arg Glu Glu 695  - #               700  - #               705 - - ACC TTG GAA TAT CGT GAT TAT GAA TTA ACG CA - #A TCA GGT AAT AGT 2875 Thr Leu Glu Tyr Arg Asp Tyr Glu Leu Thr Gl - #n Ser Gly Asn Ser 710  - #               715  - #               720 - - AAC CTA AAA GCA CAT GAT GAA TTA CAT TCA GT - #A GAA GAA ATT ATT 2920 Asn Leu Lys Ala His Asp Glu Leu His Ser Va - #l Glu Glu Ile Ile 725  - #               730  - #               735 - - GGA AGT AAT CAG AGA GAC GAA TTT AAA GGT AG - #T AAA TTC AGA GAT 2965 Gly Ser Asn Gln Arg Asp Glu Phe Lys Gly Se - #r Lys Phe Arg Asp 740  - #               745  - #               750 - - ATT TTC CAT GGT GCC GAT GGT GAT GAT CTA TT - #A AAT GGT AAT GAT 3010 Ile Phe His Gly Ala Asp Gly Asp Asp Leu Le - #u Asn Gly Asn Asp 755  - #               760  - #               765 - - GGG GAT GAT ATT CTA TAC GGT GAT AAA GGT AA - #C GAT GAG TTA AGA 3055 Gly Asp Asp Ile Leu Tyr Gly Asp Lys Gly As - #n Asp Glu Leu Arg 770  - #               775  - #               780 - - GGT GAT AAT GGT AAC GAC CAA CTT TAT GGT GG - #T GAA GGT AAT GAC 3100 Gly Asp Asn Gly Asn Asp Gln Leu Tyr Gly Gl - #y Glu Gly Asn Asp 785  - #               790  - #               795 - - AAA CTA TTA GGA GGT AAT GGC AAT AAT TAC CT - #C AGT GGT GGT GAT 3145 Lys Leu Leu Gly Gly Asn Gly Asn Asn Tyr Le - #u Ser Gly Gly Asp 800  - #               805  - #               810 - - GGC AAT GAT GAG CTT CAA GTC TTA GGC AAA TG - #G TTT TTA ATG TGC 3190 Gly Asn Asp Glu Leu Gln Val Leu Gly Lys Tr - #p Phe Leu Met Cys 815  - #               820  - #               825 - - TTC GTG GCG GTA AAG GCG ATG ATA AAC TTT AT - #G GTA GCT CAG GTT 3235 Phe Val Ala Val Lys Ala Met Ile Asn Phe Me - #t Val Ala Gln Val 830  - #               835  - #               840 - - CTG ATT TAC CTT GAT GGT GGA GAA GGT AAT GA - #T TAT CTA GAA GGA 3280 Leu Ile Tyr Leu Asp Gly Gly Glu Gly Asn As - #p Tyr Leu Glu Gly 845  - #               850  - #               855 - - GGC GAT GGT AGC GAT TTT TAT GTT TAC TGT TC - #C ACT TCA GGT AAT 3325 Gly Asp Gly Ser Asp Phe Tyr Val Tyr Cys Se - #r Thr Ser Gly Asn 860  - #               865  - #               870 - - CAT ACT ATT TAT GAT CAA GGT AAA TCT AGT GA - #T TTA GAT AAA CTA 3370 His Thr Ile Tyr Asp Gln Gly Lys Ser Ser As - #p Leu Asp Lys Leu 875  - #               880  - #               885 - - TAT TTG TCT GAT TTT TCC TTC GAT CGT CTT CT - #T GTT GAG AAA GTT 3415 Tyr Leu Ser Asp Phe Ser Phe Asp Arg Leu Le - #u Val Glu Lys Val 890  - #               895  - #               900 - - GAT GAT AAC CTT GTA CTT AGA AGT AAT GAA AG - #T AGT CAT AAT AAT 3460 Asp Asp Asn Leu Val Leu Arg Ser Asn Glu Se - #r Ser His Asn Asn 905  - #               910  - #               915 - - GGA GTA CTC ACA ATC AAA GAC TGG TTT AAA GA - #A GGG AAT AAA TAT 3505 Gly Val Leu Thr Ile Lys Asp Trp Phe Lys Gl - #u Gly Asn Lys Tyr 920  - #               925  - #               930 - - AAC CAT AAA ATT GAA CAA ATT GTT GAT AAA AA - #T GGT AGA AAA TTG 3550 Asn His Lys Ile Glu Gln Ile Val Asp Lys As - #n Gly Arg Lys Leu 935  - #               940  - #               945 - - ACA GCA GAG AAT TTA GGA ACT TAT TTC AAA AA - #T GCT CCA AAA GCT 3595 Thr Ala Glu Asn Leu Gly Thr Tyr Phe Lys As - #n Ala Pro Lys Ala 950  - #               955  - #               960 - - GAC AAT TTG CTT AAT TAT GCA ACT AAA GAA GA - #T CAG AAT GAA AGC 3640 Asp Asn Leu Leu Asn Tyr Ala Thr Lys Glu As - #p Gln Asn Glu Ser 965  - #               870  - #               975 - - AAT TTA TCT TCA CTT AAA ACT GAA TTA AGT AA - #A ATT ATT ACT AAT 3685 Asn Leu Ser Ser Leu Lys Thr Glu Leu Ser Ly - #s Ile Ile Thr Asn 980  - #               985  - #               990 - - GCA GGT AAT TTT GGT GTG GCA AAA CAA GGT AA - #T ACT GGA ATC AAT 3730 Ala Gly Asn Phe Gly Val Ala Lys Gln Gly As - #n Thr Gly Ile Asn 995  - #              1000   - #             1005 - - ACA GCT GCC TTG AAC AAT GAA GTG AAT AAA AT - #C ATT TCT TCT GCT 3775 Thr Ala Ala Leu Asn Asn Glu Val Asn Lys Il - #e Ile Ser Ser Ala 1010  - #              1015   - #             1020 - - AAT ACC TTT GCT ACT TCA CAA TTG GGT GGC TC - #A GGG ATG GGA ACA 3820 Asn Thr Phe Ala Thr Ser Gln Leu Gly Gly Se - #r Gly Met Gly Thr 1025  - #              1030   - #             1035 - - TTA CCA TCA ACG AAT GTA AAT TCA ATG ATG CT - #A GGT AAC CTA GCT 3865 Leu Pro Ser Thr Asn Val Asn Ser Met Met Le - #u Gly Asn Leu Ala 1040  - #              1045   - #             1050 - - AGA GCA GCT TAA TCA TCTGCATAAT CAATAGCAAT    - #                  - #     3900 Arg Ala Ala - - CCTATGGCTA TTCTAGGATT GCTATTTTAT TTATGGAGTC ACAAATGCCT TT - #TAACGA 3960 - - AAATAGATTA CGGATTACAT GCATTGGTAA TTCTCGCGCA ATATCACAAT GT - #TGCCGT 4020 - - ACCCTGAAGA GGTAAAACAT AAATTTGATC TTGATGGCAA AGGATTGGAT CT - #TGTTGC 4080 - - GGTTATTAGC AGCAAAATCA TTAGAATTAA AAGCCAAACG AGTAAAAAAG AG - #TATTGA 4140 - - GTTTACCATT TATTCATCTT CCTGCTTTAA TCTGGCGAGA TGATGGTCAA  - # 4190 __________________________________________________________________________

## Claims

1. A purified DNA nucleotide sequence comprising: cytolytic protein of Actinobacillus pleuropneumoniae; or pleuropneumoniae, the amino acid sequence of which is set forth as SEQ ID NO's 1 or 3.

2. The nucleotide sequence of claim 1, further comprising a nucleotide sequence encoding an activator protein, flanking at the 5' end the structural gene set forth as SEQ ID NO's 1 or 2.

3. The nucleotide sequence of claim 2, further comprising a nucleotide sequence encoding a transport protein, flanking at the 3' end the structural gene set forth as SEQ ID NO'S 1 or 2.

4. A vector comprising the nucleotide sequence of claim 1 linked to a promotor.

5. A vector according to claim 4, said nucleotide sequence being linked to an enhancer.

6. A recombinant host cell containing the nucleotide sequence of claim 1 and capable of expressing the polypeptide encoded by said nucleotide sequence.