# Patent

## Title
Protein fragments of virB10 and sero-detection of anaplasma phagocytophium

## Abstract

Disclosed are cloning and expression of a plurality of protein fragments of virB10, a Type IV Secretion System (TIVSS) in Anaplasma phagocytophilum. Such recombinant protein fragments are useful in the ELISA detection of anaplasma pathogen. The use of same as kits for ELISA is also disclosed.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Applications No. 61/208,761 filed Feb. 27, 2009, the contents of which are incorporated by reference herein in their entirety.


FIELD OF THE INVENTION
The present invention generally relates to the field of diagnostic assays for the detection of infectious agents in an animal, including humans. Particular embodiments disclosed herein encompass protein fragments of virB10 (a Type IV Secretion Protein System) (TIVSS) that are useful in the sero-detection of Anaplasma phagocytophilum. 
BACKGROUND OF THE INVENTION
Anaplasma phagocytophilum is a tick-borne pathogen responsible for granulocytic anaplasmosis in humans (Bakken J. S., et al.: Human granulocytic ehrlichiosis in the upper Midwest United States. A new species emerging? JAMA 272: 212-218, 1994). There has been a steady rise in cases of anaplasma infections, alone or through co-infection with other tick-borne pathogens (Varde S., et al.: Prevalence of tick-borne pathogens in Ixodes scapularis in a rural New Jersey County. Emerg. Infect. Dis. 4: 97-99, 1998). Left unchecked, anaplasma infection can be a potentially fatal disease resulting from the targeting and replication of Ap within human neutrophils (Bakken J. S. et al.: JAMA 272: 212-218, 1994). Anaplasma phagocytophilum infection thus emerges as a significant healthcare concern.
Detection of anaplasma infection is crucial. Ideally, a diagnostic assay should be capable of detecting anaplasma infection at its earliest stages, when antibiotic treatment is most effective and beneficial. Traditional detection methods for anaplasma infection includes: (i) microscopic identification of morulae in granulocytes, (ii) PCR analysis using whole blood, (iii) isolation of the anaplasma bacterium from whole blood, and (iv) serological tests, particularly indirect immunofluorescence assay (IFA). Microscopic examination is tedious and prone to sampling error. PCR test is sensitive in detecting the tick-borne pathogen during the period of time when the pathogen is present in the blood of infected patients. IFA is most commonly used (Park, J., et al.: Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays. Clinical and Diagnostic Laboratory Immunology 10(6): 1059-1064, 2003), but this test often gives false positive results. Such results can be attributed in part to the use of whole-cell antigens because such proteins may be shared with other bacteria (Magnarelli, L. A., et al.: Use of recombinant antigens of Borrelia burgdorferi and Anaplasma phagocytophilum in enzyme-linked immunosorbent assays to detect antibodies in white-tailed deer. J. Wildlife Dis. 40(2): 249-258, 2004). When clinical symptoms are manifested or high and stable antibody titers to Anaplasma phagocytophilum are found in patient blood, it reaches a late infection stage and bypass the window of antibiotic treatment.
So far, there are only a few surface proteins on anaplasma pathogen that are used in diagnostic assay for immuno-responses (i.e., IgG and IgM responses). It is generally believed that outer membrane proteins in pathogens are target for eliciting an immuno-response because they may be the first to be exposed to immune cells of a host. Regarding the anaplasma phagocytophilum species, U.S. Pat. No. 6,964,855 discloses the use of an outer membrane protein and its fragments in a detection assay. U.S. Pat. No. 7,304,139 discloses a major surface protein 5 (MSP5) and its use in a diagnostic test. The '139 patent discloses a few patient's reactivity towards MSP5 and it lacks any data relating sensitivity and specificity, let alone any IgG/IgM distinction. Zhi et al. discloses cloning and expression of an outer membrane protein of 44 kDa and its use in a Western immunoblot assay (J. Clinical Microbiology 36(6): 1666-1673, 1998). Both MSP5 and p44 are outer membrane proteins in Anaplasma phagocytophilum. To the best knowledge of the inventors, there is no report on using any intracellular protein as an antigenic protein, let alone its possible use in ELISA detection for Anaplasma phagocytophilum. 
In Agrobacterium tumefaciens, TIVSS consists of twelve (12) protein components. virB5 and a part of virB2 are proteins located on the outer surface of the pathogen. On the other hand, the rest of the TIVSS in Agrobacterium tumefaciens reside within the pathogen (See, FIG. 1). TIVSS in Agrobacterium tumefaciens may represent a prototype for TIVSS in other species. The number of TIVSS protein components varies among various different species in the family. TIVSS in Agrobacterium tumefaciens is believed to form a conduit for transportation of macromolecules (such as proteins) across the cell membrane. Anaplasma phagocytophilum is a phylogenetically distant species. TIVSS in Anaplasma phagocytophilum consists of eight (8) protein components. And the manner by which TIVSS proteins assembly and their respective functions in Anaplasma phagocytophilum is presently unknown. Flabio R. Araujo et al. recently reported that sera of cattle infected with Anaplasma marginale (a phylogenetically distant species of Anaplasma phagocytophilum) can recognize recombinant virB9, virB10, and elongation factor-Tu (EF-Tu). To the best of the inventor's knowledge, there is no information exists regarding the cloning and recombinant expression of the TIVSS protein components in Anaplasma phagocytophilum. 
There is a continuing need in the discovery of a novel antigen present in Anaplasma phagocytophilum that may be useful in sero-detection of this pathogen. The present invention cures all the above-mentioned defects and provides a useful detection assay for Anaplasma phagocytophilum infection. Disclosed herein are the cloning, expression, purification, and use of a recombinant type IV secretion system (TIVSS) protein virB10 (rTIVSS virB10) and its protein fragments. Particular embodiments include the development of a diagnostic ELISA test useful for detecting IgM/IgG antibody responses to Anaplasma phagocytophilum. The present assay utilizes recombinant virB10 protein fragments and the data show that they can be used to discriminate Anaplasma phagocytophilum IFA-positive and IFA-negative patient samples with high sensitivity and specificity.
SUMMARY OF THE INVENTION
The present invention provides polypeptides of Anaplasma phagocytophilum that are useful in the detection of Anaplasma phagocytophilum. The present invention provides recombinant TIVSS protein fragments and methods of using these polypeptides in the detection of infections with Anaplasma phagocytophilum, which can be useful in the diagnosis of human granulocytic anaplasmosis.
In one aspect, the present invention provides an isolated polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 15.
In another aspect, the present invention provides an isolated polynucleotide with nucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16.
In one aspect, the present invention provides a vector comprising the isolated polynucleotides of virB10 protein fragments. virB10 protein fragments may include virB fragments 1-5. Preferably, the vector comprises the isolated polynucleotide with nucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16. The vector may be pET. The vector may further comprise a promoter of DNA transcription operably linked to the isolated polynucleotides of interest. The vector may further comprises a promoter of DNA transcription operably linked to the isolated isolated polynucleotides of interest. The vector may be pET, pENTR, or pCR®8/GW/TOPO®. The promoter may be a lac promoter, trp promoter or tac promoter.
In one aspect, the present invention provides a host cell comprising the vector. The host cell may be E. coli and the E. coli may include NOVABLUE K12 strain or BL21 (DE3).
In one aspect, the present invention provides a method of producing an isolated polypeptide of virB10 fragments having an amino acid set forth in SEQ ID NO: 13 or SEQ ID NO: 15. The method comprises the steps of: (i) introducing the isolated virB10 gene fragments into a host cell; (ii) growing the host cell in a culture under suitable conditions to permit production of said isolated polypeptide; and (iii) isolating the isolated polypeptide of virB10. Preferably, the virB10 gene fragments include isolated polynucleotide with nucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 16.
In one aspect, the present invention provides a method of detecting the presence of an antibody against Anaplasma phagocytophilum in a biological sample of a mammal, comprising: (i) immobilizing an isolated polypeptide of virB10 fragments onto a surface, the amino acid sequences of virB10 are set forth in SEQ ID NO: 13 or SEQ ID NO: 15; (ii) contacting the isolated polypeptide with a patient's biological sample, under conditions that allow formation of an antibody-antigen complex between the immobilized polypeptide (antigen) and an antibody against Anaplasma phagocytophilum; and (iii) detecting the formation of the antibody-antigen complex; the detected antibody-antigen complex is indicative of the presence of said antibody against Anaplasma phagocytophilum in the biological sample. Preferably, the mammal is a human. ELISA test employs an IgG or IgM assay. Preferably, the ELISA has a sensitivity of at least >70%, and a specificity of at least >70%.
In another aspect, the present invention provides a method of diagnosing an infection of Anaplasma phagocytophilum in a mammal, comprising the steps of: (i) obtaining a biological sample from a mammal suspected of having an Anaplasma phagocytophilum infection; (ii) immobilizing an isolated polypeptide of virB10 protein fragments onto a surface, the amino acid sequences of virB10 protein fragments are set forth in SEQ ID NO: 13 or SEQ ID NO: 15; (iii) contacting the immobilized polypeptide with the biological sample, under conditions that allow formation of an antibody-antigen complex; and (iv) detecting said antibody-antigen complex. The detected antibody-antigen complex is indicative of the presence of said antibody against Anaplasma phagocytophilum in the biological sample. Preferably, the biological sample is whole blood, and the antibody is IgG or IgM.
In yet another aspect, the present invention provides an article of manufacture comprising a packaging material; and the isolated polypeptides of virB10 protein fragments. The article of manufacture may further comprise an instruction for detecting the presence of antibody against Anaplasma phagocytophilum. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically depicts the Agrobacterium tumefaciens Type IV Secretion System (TIVSS). Modified from KEGG: Kyoto Encyclopedia of Genes and Genomes (www.genome.ad.jp/dbgetbin/get_pathway?org_name=aph&mapno=03080).
FIG. 2 depicts the EK/LIC PCR Amplification of Anaplasma Genes Encoding TIVSS proteins of Anaplasma phagocytophilum. Lane 13 depicts the full length virB10 gene (1,305 bp).
FIG. 3 depicts the Post-PCR Clean-Up of Anaplasma Clones for Recombinant Expression. The arrow in this figure shows the virB10 amplicon.
FIG. 4 depicts the pET-30 Vector Containing full-length virB10 Gene.
FIG. 5 depicts the Nucleotide Sequence for TIVSS virB10 Gene in Anaplasma phagocytophilum (accession #YP—505896) (SEQ ID NO: 10), and the deduced amino acid sequence of TIVSS virB10 protein (SEQ ID NO: 11).
FIG. 6 depicts the Colony PCR of virB10 Transformants in NOVABLUE E. coli. 
FIG. 7 depicts the Colony PCR of virB10 Transformants in BL21 (DE3) E. coli. 
FIG. 8 depicts the protocol for IPTG-Induced Recombinant TIVSS Protein (i.e., virB10) Expression in BL21 E. coli. 
FIG. 9 depicts the IPTG Induction of TIVSS Proteins (including virB10) (Soluble v. Insoluble Fractions).
FIG. 10 depicts the Ni-NTA Purification of 6× His-Tagged Recombinant TIVSS virB10 Protein.
FIG. 11 depicts the IgM and IgG ELISA for Recombinant virB10 of Anaplasma phagocytophilum. 
FIG. 11a depicts the ROC analysis for Recombinant virB10 IgM ELISA.
FIG. 12 depicts the Location of Fragments 1-5 relative to the Full-Length virB10 protein.
FIG. 13 depicts the Nucleotide Sequence for TIVSS virB10 Fragment 1 (SEQ ID NO: 12), and the Deduced Amino Acid Sequence of TIVSS virB10 Fragment 1 (SEQ ID NO: 13).
FIG. 14 depicts the Nucleotide Sequence for TIVSS virB10 Fragment 2 (SEQ ID NO: 14), and the Deduced Amino Acid Sequence of TIVSS virB10 Fragment 2 (SEQ ID NO: 15).
FIG. 15 depicts the Nucleotide Sequence for TIVSS virB10 Fragment 3 (SEQ ID NO: 16), and the Deduced Amino Acid Sequence of TIVSS virB10 Fragment 3 (SEQ ID NO: 17).
FIG. 16 depicts the Nucleotide Sequence for TIVSS virB10 Fragment 4 (SEQ ID NO: 18), and the Deduced Amino Acid Sequence of TIVSS virB10 Fragment 4 (SEQ ID NO: 19).
FIG. 17 depicts the Nucleotide Sequence for TIVSS virB10 Fragment 5 (SEQ ID NO: 20), and the Deduced Amino Acid Sequence of TIVSS virB10 Fragment 5 (SEQ ID NO: 21).
FIG. 18 depicts the Antigenicity Plot of virB10 and the Location of Fragments 1-5 (Shown Below the Plot) Relative to the Antigenic Profile of Full Length virB10 Protein.
FIG. 19 depicts the EK/LIC PCR Amplification of Anaplasma TIVSS virB10 Fragments.
FIG. 20 depicts the Colony PCR of Fragments 1-3 Transformants in NOVABLUE E. coli. 
FIG. 21 depicts the Colony PCR of Fragment 4 Transformants in NOVABLUE E. coli. 
FIG. 22 depicts the pET-30 Vector Containing virB10 Gene Fragments.
FIG. 23 depicts the Presence of Fragments 1 and 2 in the Soluble Fraction following Induction of Expression.
FIG. 24 depicts the COOMASSIE-Stained Gel and His-Tag Western Blot of Fragments 1 and 2.
FIG. 25 depicts the Nickel Column Purification of Fragments 1 and 2.
FIG. 26 depicts the Induction of Fragments 3 and 4. This figure shows the Presence of Fragments 3 and 4 in the Insoluble Fraction.
FIG. 27 depicts the Purification of the Inclusion Body Fraction
FIG. 28 depicts the Nickel Column Purification of Fragments 3 and 4 from the Inclusion Body Fraction.
FIG. 29 depicts the COOMASSIE-Stained Gel and His-Tag Western Blot of Fragments 3 and 4.
FIG. 30 depicts the Induction of Fragment 5. The arrow shows the presence of the Induced protein in the Insoluble (Inclusion Body) Fraction.
FIG. 31 depicts the Nickel Column Purification of Fragment 5.
FIG. 32 depicts the His-Tag Western Blot of Fragment 5.
FIG. 33 depicts the IgG ELISA for Recombinant virB10 Fragments 1 and 2.
FIG. 34 depicts the IgM ELISA for Recombinant virB10 Fragments 1 and 2.
FIG. 35 depicts the IgM ELISA Analysis for Combined virB10 Fragments 1 and 2.
FIG. 36 depicts the IgG ELISA Analysis for Combined virB10 Fragments 1 and 2.
FIG. 37 depicts the IgG ELISA for Recombinant virB10 Fragments 3 and 4.
FIG. 38 depicts the IgG ELISA for Recombinant virB10 Fragment 5.



DETAILED DESCRIPTION OF THE INVENTION
The present invention can be better understood from the following description of preferred embodiments, taken in conjunction with the accompanying drawings. It should be apparent to those skilled in the art that the described embodiments of the present invention provided herein are merely exemplary and illustrative and not limiting.
DEFINITIONS
Various terms used throughout this specification shall have the definitions set out herein.
As used herein, “virB10” refers to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 26 (NCBI Accession No. YP—505896). The polypeptide represents the type IV secretion system virB10 protein present in Anaplasma phagocytophilum strain HZ. The virB10 polypeptide is shown by the present inventors to bind to antibodies that are present in Anaplasma patients' sera in an ELISA assay.
As used herein, “virB10 fragments” refers to protein fragments of the full length virB10 polypeptide. The term “virB10 fragment” is intended to include at least the five (5) protein fragments of virB10 disclosed herein in this application (namely, fragment 1, fragment 2, fragment 3, fragment 4, and fragment 5). The amino acid sequences of the virB10 protein fragments are set forth below: (i) virB10 protein fragment 1 having amino acid as set forth in SEQ ID No: 13, (ii) virB10 protein fragment 2 having amino acid as set forth in SEQ ID NO: 15, (iii) virB10 protein fragment 3 having amino acid as set forth in SEQ ID NO: 17, (iv) virB10 protein fragment 4 having amino acid as set forth in SEQ ID NO: 19, and (v) virB10 protein fragment 5 having amino acid as set forth in SEQ ID NO: 21. One of ordinary skill in the art would appreciate that the virB10 protein fragments would encompass protein fragment variants (e.g., conservative substitutions of amino acids) insofar as the protein fragments still possess the ability to bind to IFA(+) sera from Anaplasma infected patients' sera in an ELISA assay.
As used herein, the term “ELISA” refers to “Enzyme-Linked ImmunoSorbent Assay” and is a biochemical technique used in detecting the presence of antibody or antigen in a sample.
As used herein, the term “IFA” refers to immunofluorescence assay. “IFA sero-positive sera from a patient” refers to sera (obtained from a patient) that exhibit positive immunofluorescence staining towards cells that have been infected with Anaplasma phagocytophilum. “IFA sero-negative sera from a patient” refers to sera (obtained from a patient) that exhibit negligible immunofluorescence staining towards cells that have been infected with Anaplasma phagocytophilum. 
As used herein, the terms “polypeptide,” “peptide,” or “protein” are used interchangeably.
As used herein, the term “recombinant polypeptide” refers to a polypeptide that is recombinantly expressed by a host cell via the use of a vector that has been modified by the introduction of a heterologous nucleic acid. For purposes of the present invention, these polypeptides are intended to encompass some polypeptide variations insofar as they retain the ability to bind to antibodies present in Anaplasma infected patients in an ELISA assay with comparable sensitivity and specificity. One of an ordinary skill in the art would appreciate that the polypeptide variations may include (i) conservative substitutions, (ii) substitution, (iii) addition, and (iv) deletion of amino acids. It would be further appreciated that a polypeptide variant having a sufficiently high % amino acid sequence identity (e.g., >95%), when exhibited similar antibody binding activity as to the parent polypeptide, is intended to be encompassed by the present invention.
As used herein, the term “% amino acid sequence identity” is defined as the percentage of amino acid residues that are identical to the amino acid residues in the TIVSS (e.g., virB10) polypeptide or protein fragments thereof. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are well within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
As used herein, the term “mammal” refers to any vertebrate of the class mammalia, having the body more or less covered with hair, nourishing the young with milk from the mammary glands, and, with the exception of the egg-laying monotremes, giving birth to live young. Preferably, the mammal is human.
As used herein, the term “primer” refers to a nucleotide sequence which can be extended by template-directed polymerization. For the purpose of this application, the term “nucleotide sequence” is intended to include DNA or modification thereof.
As used herein, the term “biological sample” may include but are not limited to blood (e.g., whole blood, blood serum, etc.), cerebrospinal fluid, synovial fluid, and the like from a mammal such as a human or domestic animal. Extraction of nucleic acids from biological samples is known to those of skill in the art.
As used herein, the term “ROC” refers to Receiver Operating Characteristics Analysis. ROC analysis is a standard statistical tool for evaluation of clinical tests. ROC accesses the performance of the system in terms of “Sensitivity” and “1-Specificity” for each observed value of the discriminator variable assumed as decision threshold (i.e., cutoff value to differentiate between two groups of response). For ELISA, the cutoff value can be shifted over a range of observed values (i.e., OD450 nm reading), and Sensitivity and 1-Specificity can be established for each of these values. The optimal pair of Sensitivity and Specificity is the point with the greatest distance in a Northwest direction.
The present invention provides recombinant and synthetic polypeptides that, when assayed in an ELISA assay, react to IFA sero-positive sera and do not react to IFA sero-negative sera from a patient infected with Anaplasma phagocytophilum. 
Recombinant Polypeptides of TIVSS
The present invention specifically contemplates expression and preparation of recombinant and synthetic polypeptides of virB10 and protein fragments thereof, characterized by being capable of binding to antibodies present in IFA positive patient sera. In one embodiment, the present invention thus comprises the isolated nucleic acid having the nucleotide sequence set forth in FIG. 5 (SEQ ID NO: 10). The recombinant proteins of virB10 expressed by the nucleic acids described herein encompasses the protein set forth in FIG. 5 (SEQ ID NO: 11). The recombinant virB10 protein described herein possesses the ability to bind to antibodies present in IFA positive sera (and not IFA negative sera).
In another embodiment, the present invention thus comprises the isolated nucleic acid having the nucleotide sequence set forth in FIG. 13 (SEQ ID NO: 12). The recombinant proteins expressed by the nucleic acids described herein encompasses those proteins set forth in FIG. 13 (SEQ ID NO: 13).
In another embodiment, the present invention thus comprises the isolated nucleic acid having the nucleotide sequence set forth in FIG. 14 (SEQ ID NO: 14). The recombinant proteins expressed by the nucleic acids described herein encompasses those proteins set forth in FIG. 14 (SEQ ID NO: 15).
In another embodiment, the present invention thus comprises the isolated nucleic acid having the nucleotide sequence set forth in FIG. 15 (SEQ ID NO: 16). The recombinant proteins expressed by the nucleic acids described herein encompasses those proteins set forth in FIG. 14 (SEQ ID NO: 17).
In another embodiment, the present invention thus comprises the isolated nucleic acid having the nucleotide sequence set forth in FIG. 16 (SEQ ID NO: 18). The recombinant proteins expressed by the nucleic acids described herein encompasses those proteins set forth in FIG. 14 (SEQ ID NO: 19).
In another embodiment, the present invention thus comprises the isolated nucleic acid having the nucleotide sequence set forth in FIG. 17 (SEQ ID NO: 20). The recombinant proteins expressed by the nucleic acids described herein encompasses those proteins set forth in FIG. 14 (SEQ ID NO: 21).
The virB11 protein fragments described herein possess the ability to bind to antibodies present in IFA positive sera (and not IFA negative sera).
In one embodiment, the present invention provides a recombinant polypeptide containing an amino acid sequence as set forth in SEQ ID NO: 13. In another embodiment, the present provides a recombinant polypeptide containing an amino acid sequence set forth in SEQ ID NO: 15.
It is understood that these recombinant polypeptides encompass variants. One type of variants includes modification of amino acids of recombinant polypeptides; such as, for example, substitution, deletion, or addition of amino acids. The present invention is intended to encompass the polypeptide variants of virB10 and virB11 that retain the antibody binding ability towards IFA sero-positive sera and do not react to IFA sero-negative sera from Anaplasma infected patients. One of ordinary skill in the art would recognize that conservative amino acid substitutions may include simply substituting glutamic acid with aspartic acid; substituting isoleucine with leucine; substituting glycine or valine, or any divergent amino acid, with alanine, substituting arginine or lysine with histidine, and substituting tyrosine and/or phenylalanine with tryptophan. In another embodiment, addition and deletion of single amino acid may be employed. It is also appreciated by one of ordinary skill in the art that a few amino acids can be included or deleted from each or both ends, or from the interior of the polypeptide without significantly altering the peptide's ability to bind antibody (i.e., maintain high sensitivity and specificity (>70%), when tested in an ELISA assay.
Recombinant Expression of virB10 and virB11 Polypeptides: Vectors and Hosts
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
A DNA sequence is “operatively linked” or “operably linked” to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted upstream (5′) of and in reading frame with the gene. A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
In one embodiment, the present invention provides the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be recombinantly expressed by operatively linking the sequences to an expression control sequence in an appropriate expression vector; and expressing that linked vector via transformation in an appropriate unicellular host. Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and Synthetic DNA sequences. Suitable vectors include pET, pENTR, and pCR®8/GW/TOPO® and the like. The promoter contains lac promoter, tip promoter and tac promoter.
In one embodiment, a host cell contains the vector comprising the polynucleotides of the present invention. Exemplary host cell includes E. coli. Various E. coli strains include, for example, NOVABLUE strain, BL21 (DE3) or BL21 pLsS (DE3).
It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired expression without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered. In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly as regards potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. Considering these and other factors, a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture.
For recombinant expression of the various proteins used in this application, genes encoding the various proteins of interest can be conveniently inserted into a cloning vector and the vector containing the gene of interest is transfected or transformed into a suitable host cell for protein expression. Various publicly available vectors may be used. For example, vectors may include a plasmid, cosmid, viral particle, or phage. Examples of vectors included pET30®, pENTR®, pCR8/GW/TOPO® and the like. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, a marker gene, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components as well as the gene of interest employs standard ligation techniques which are known to the skilled artisan.
Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. 
Examples of suitable selectable markers for mammalian cells include those that enable the identification of cells competent to take up the antigen-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).
A number of promoters can be used in order to enhance the expression of the gene of interest. In one embodiment, a promoter can be employed which will direct expression of a polynucleotide of the present invention in E. coli. Other equivalent transcription promoters from various sources are known to those of skill in the art. Exemplary promoters include the β-lactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978)), alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980)), and the like.
A promoter may be operably linked to the protein-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. For example, promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the protein of interest.
Transcription of a DNA encoding the antigen by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that can act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′ or 3′ to the 15-kDa coding sequence, but is preferably located at a site 5′ from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding Anaplasma phagocytophilum antigen.
The nucleic acid (e.g., genomic DNA) encoding recombinant Anaplasma phagocytophilum antigen of the present invention may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. For example, a type IV secretion system (TIVSS) protein, such as full-length virB10 (SEQ ID No.10) may be inserted into a replicable vector for cloning and for expression of full-length virB9 protein or fragments thereof. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art.
Host cells are transfected or transformed with expression or cloning vectors described herein for antigen production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991).
Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting.
Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, Ca2PO4, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., or electroporation is generally used for prokaryotes. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, See Keown et al., Methods in Enzymology, 185:527-537 (1990). The particular selection of host/cloning vehicle combination may be made by those of skill in the art after due consideration of the principles set forth without departing from the scope of this invention (See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition, 1989, Cold Spring Harbor Press, NY).
The antigen may be recombinantly produced as a fusion polypeptide with a heterologous polypeptide. The heterologous polypeptide may serve as a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the antigen-encoding DNA that is inserted into the vector. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. An overview of expression of recombinant proteins is found in Methods of Enzymology v. 185, Goeddel, D. V. ed. Academic Press (1990).
Recombinant gene expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Recombinant gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to Anaplasma phagocytophilum DNA and encoding a specific antibody epitope.
After expression, recombinant antigen may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. TRITON-X 100) or by enzymatic cleavage. Cells employed in expression of Anaplasm phagocytophilum antigen can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.
It may be desired to purify recombinant antigen from host cell proteins. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; metal chelating columns to bind epitope-tagged forms of the protein of interest. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular antigen produced.
ELISA Assay
Detection of antibody binding in IFA sero-positive sera may be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay), western blots, and the like. In one embodiment, antibody binding is assessed by detecting a label on the primary antibody. In another embodiment, the primary antibody is assessed by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select specific epitopes of recombinant or synthetic polypeptide, one may assay antibody binding in an ELISA assay wherein the polypeptides or its fragments containing such epitope.
As appreciated by one skilled in the art, an enzyme-linked immunosorbent assay (ELISA) may be employed to detect antibody binding in IFA sero-positive sera. In an initial step of an ELISA, an antigen is immobilized onto a surface (for example by passive adsorption known as coating). For purposes of this application, exemplary antigens include Anaplasma phagocytophilum type IV secretion system proteins (eg. virB10 and virB11), hemolysin, succinate dehydrogenase and p44-8 outer membrane protein and the like. Recombinant full-length protein as well as fragments thereof may be used. Immobilization of antigen may be performed on any inert support that is useful in immunological assays. Examples of commonly used supports include small sheets, SEPHADEX and assay plates manufactured from polyethylene, polypropylene or polystyrene. In a preferred embodiment the immobilized antigens are coated on a microtiter plate that allows analysis of several samples at one time. More preferably, the microtiter plate is a microtest 96-well ELISA plate, such as those sold under the name NUNC MAXISORP or IMMULON.
Antigen immobilization is often conducted in the presence of a buffer at an optimum time and temperature optimized by one skilled in the art. Suitable buffers should enhance immobilization without affecting the antigen binding properties. Sodium carbonate buffer (e.g., 50 mM, pH 9.6) is a representative suitable buffer, but others such as Tris-HCl buffer (20 mM, pH 8.5), phosphate-buffered saline (PBS) (10 mM, pH 7.2-7.4) are also used. Optimal coating buffer pH will be dependent on the antigen(s) being immobilized. Optimal results may be obtained when a buffer with pH value 1-2 units higher than the isoelectric point (pI) value of the protein is used. Incubation time ranges from 2-8 hours to overnight. Incubation may be performed at temperatures ranging from 4-37° C. Preferably, immobilization takes place overnight at 4° C. The plates may be stacked and coated long in advance of the assay itself, and then the assay can be carried out simultaneously on several samples in a manual, semi-automatic, or automatic fashion, such as by using robotics.
Blocking agents are used to eliminate non-specific binding sites in order to prevent unwanted binding of non-specific antibody to the plate. Examples of appropriate blocking agents include detergents (for example, TWEEN-20, TWEEN-80, TRITON-X 100, sodium dodecyl sulfate), gelatin, bovine serum albumin (BSA), egg albumin, casein, non-fat dried milk and the like. Preferably, the blocking agent is BSA. Concentrations of blocking agent may easily be optimized (e.g. BSA at 1-5%). The blocking treatment typically takes place under conditions of ambient temperatures for about 1-4 hours, preferably 1.5 to 3 hours.
After coating and blocking, sera from the control (IFA sero-negative) or IFA sero-positive patients are added to the immobilized antigens in the plate. Biological sample (i.e., sera) may be diluted in buffer. Phosphate Buffered Saline (PBS) containing 0.5% BSA, 0.05% TWEEN 20® detergent may be used. TWEEN 20® acts as a detergent to reduce non-specific binding.
The conditions for incubation of the biological sample and immobilized antigen are selected to maximize sensitivity of the assay and to minimize dissociation. Preferably, the incubation is accomplished at a constant temperature, ranging from about 0° C. to about 40° C., preferably from about 22 to 25° C. to obtain a less variable, lower coefficient of variant (CV) than at, for example, room temperature. The time for incubation depends primarily on the temperature, being generally no greater than about 10 hours to avoid an insensitive assay. Preferably, the incubation time is from about 0.5 to 3 hours, and more preferably 1.5-3 hours at room temperature to maximize binding to immobilized capture antigen.
Following incubation of the biological sample and immobilized antigen, unbound biological sample is separated from the immobilized antigen by washing. The solution used for washing is generally a buffer (“washing buffer”) with a pH determined using the considerations and buffers described above for the incubation step, with a preferable pH range of about 6-9. Preferably, pH is 7. The washing may be done three or more times. The temperature of washing is generally from refrigerator to moderate temperatures, with a constant temperature maintained during the assay period, typically from about 0-40° C., more preferably about 4-30° C. For example, the wash buffer can be placed in ice at 4° C. in a reservoir before the washing, and a plate washer can be utilized for this step.
Next, the immobilized capture antigen and biological sample are contacted with a detectable antibody at a time and temperature optimized by one skilled in the art. Detectable antibody may include a monoclonal antibody or a polyclonal antibody. These antibodies may be directly or indirectly conjugated to a label. Suitable labels include moieties that may be detected directly, such as fluorochrome, radioactive labels, and enzymes, that must be reacted or derivatized to be detected. Examples of such labels include the radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, horseradish peroxidase (HRP), alkaline phosphatase, and the like. Preferably, the detection antibody is a goat anti-human IgG polyclonal antibody that binds to human IgG and is directly conjugated to HRP. Incubation time ranges from 30 minutes to overnight, preferably about 60 minutes. Incubation temperature ranges from about 20-40° C., preferably about 22-25° C., with the temperature and time for contacting the two being dependent on the detection means employed.
The conjugation of such labels to the antibody, including the enzymes, is a standard manipulative procedure for one of ordinary skill in immunoassay techniques. See, for example, O'Sullivan et al. “Methods for the Preparation of Enzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods in Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic Press, New York, N.Y., 1981), pp. 147-166.
Because IgG may occasionally interfere in IgM detection assays, IgG in patient sera may be removed prior to IgM ELISA. Ideally, an anti-human IgG antibody is used to neutralize the IgG in human sera. Commercial reagents such as GullSORBTM (Meridian Bioscience, Inc., Cincinnati, Ohio) may be used. The method for IgG removal can be conveniently optimized by one of ordinary skill in the art. For example, human sera can be incubated with anti-human IgG antibody prior to the IgM ELISA assay.
Diagnostic Kits Employing Recombinant virB10 Polypeptide
The present invention provides a kit for the diagnosis of anaplasma infection. In one embodiment, the kit is an ELISA kit containing recombinant polypeptides described herein, detection reagents including primary or secondary antibodies, and other necessary reagents including enzyme substrates and color reagents. Additional components that may be present within such kits include an instruction detailing the detection procedure for Anaplasma phagocytophilum, using the recombinant polypeptides of the present invention. The diagnostic kit of the present invention further comprises a positive and negative serum control. The diagnostic kit of the present invention can also be used in diagnosing other infectious diseases involving Anaplasma phagocytophilum such as Human Granulocytic Anaplasmosis (HGA).
The following Examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL STUDIES
Example 1
Type IV Secretion System in Anaplasma phagocytophilum 
FIG. 1 is a schematic depiction of the Type IV Secretion System (TIVSS) in plant pathogen Agrobacterium tumefaciens (modified from Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.ad.jp/dbgetbin/get_pathway?org_name=aph&mapno=03080). TIVSS is believed to form a conduit for transportation of macromolecules such as proteins and DNA across the cell membrane. TIVSS in Agrobacterium tumefaciens represents a prototype, albeit the protein components within the TIVSS may vary among the different pathogens. For example, while Agrobacterium spp. have twelve (12) proteins (See, FIG. 1), Anaplasma phagocytophilum (a phylogenetically distant species) contains only eight (8) proteins. Notably, virB1, virB2, virB5 and virB7 are absent in Anaplasma phagocytophilum. The exact structural organization of TIVSS in Anaplasma phagocytophilum is presently unclear.
TIVSS is essential for establishing infection in Anaplasma phagocytophilum. There is no information about the immunogenicity of the various TIVSS proteins during the anaplasma infection. So far in Anaplasma phagocytophilum, a non-TIVSS protein (p44; a surface protein, also known as p44-8) is known to induce an antibody response in a human host (Ijdo, J. W. et al., Cloning of the gene encoding the 44-kilodalton antigen of the agent of human granulocytic ehrlichiosis and characterization of the humoral response. Infection and Immunity, 66(7): 3264-3269, 1998).
The present inventors surprisingly discovered that virB10 (a TIVSS protein components) and protein fragments thereof are good candidate biomarkers for the diagnosis of Anaplasma phagocytophilum infection. Evidence is presented herein to demonstrate that recombinantly expressed virB10 and protein fragments thereof, when immobilized in an ELISA assay, are good detection marker for an IgG/IgM antibody response to Anaplasma phagocytophilum infection. Specifically, virB10 fragments xy are good antigens for ELISA assay in detecting Anaplasma phagocytophilum. 
Example 2
Cloning and Expression of virB10
I) PCR Amplification and Ligation into Plasmid Vector
We sought to determine if virB10 possesses antibody recognition sites. First we cloned and recombinantly expressed the full-length virB10 protein in Anaplasma phagocytophilum. 
Our cloning strategy involved the design and preparation of synthetic oligonucleotides ( ̃30 bp in length) and use of them in amplifying the virB10 gene. As controls, we also cloned two (2) non-TIVSS proteins (i.e., succinate dehydrogenase iron-sulfur subunit and p44 outer membrane protein) and used them for as comparison. Table 1 shows the nucleotide sequence of the various oligonucleotides (i.e., SEQ ID Nos. 1-6) used in the PCR amplification reaction.
Genomic DNA of Anaplasma phagocytophilum (a generous gift from Dr. S. Dumler at Johns Hopkins University) was used as the template for each of the PCR reactions. Synthetic oligonucleotides corresponding to the virB10 gene were used for the PCR amplification reactions. Using the synthetic oligonucleotides (sequence listed in Table 1) and genomic DNA from Anaplasma phagocytophilum, we successfully amplified the virB10 gene; as well as two (2) non-TIVSS genes (i.e., succinate dehydrogenase iron-sulfur and p44 proteins) (See, FIGS. 2 and 3).
FIG. 2 shows an agarose gel of the amplified genes prior to processing of the PCR reactions in preparation for ligation into pET30 vector. The virB10 amplicon having an expected size ( ̃1.0 kb) is shown by the arrow in this figure. In preparation for ligation with the vector, the PCR amplification reactions were treated to remove any remaining nucleotides, primers, and reaction components.
FIG. 3 shows a COOMASSIE-stained gel of the amplified genes following clean-up of the PCR reactions. The arrow in this figure shows the virB10 amplicon of expected size ( ̃1.0 Kb)
The resulting PCR products were then treated with T4 DNA polymerase and ligated into pET30 using standard protocols (See, FIG. 4). Ligation of the virB10 insert DNA (including succinate dehydrogenase iron-sulfur and p44 protein insert DNAs) was performed as described below.
II) T4 Polymerase Treatment of PCR Products and Ligation into pET30 Vector
In order to ligate the cloned insert DNA with the plasmid vector, it is necessary to create compatible ends between the amplicon and the chosen vector (e.g., pET30 Ek/LIC). We generated overhangs compatible with the Ek/LIC cloning vector on the insert DNA by T4 DNA polymerase treatment of the PCR amplicon. We ligated the treated amplicon into the expression vector to form pET30/insert DNA.
FIG. 4 depicts the pET30 vector containing the inserted gene (e.g., full-length virB10, succinate dehydrogenase iron-sulfur and p44). The nucleotide sequences of virB10, succinate dehydrogenase iron-sulfur and p44 are publicly available and their accession numbers are listed in Table 2.
III) Transformation of Recombinant Clones into NOVABLUE E. coli 
In these series of experiments, we transformed the ligated DNAs (annealing reaction) into host bacterial cells (NOVABLUE E. coli). The ligated DNA was virB10 amplicons as well as succinate dehydrogenase iron-sulfur and p44 amplicons. We chose NOVABLUE E. coli because this bacterial strain is optimized for producing a stable cell line containing a recombinant insert (see, NOVABLUE Ek/LIC manual).
Transformation into NOVABLUE competent E. coli (NOVAGEN) was performed using standard protocols. First, appropriate numbers of 20 μl aliquots of competent cells were prepared from −80° C., and allowed to thaw on ice for several minutes, followed by the addition of 1 μl of the annealing reaction and gentle stirring. The mixture was further incubated on ice for an additional 5 minutes, followed by heating the tubes for 30 seconds in a 42° C. water bath. The tubes were immediately placed on ice for 2 minutes. SOC (Super Optimal broth with Catabolite repression medium, containing 2% w/v bacto-tryptone, 0.5% w/v bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose) (at room temperature) was added into the tubes, and the reactions were further incubated for 1 hour at 37° C. with shaking (250 rpm). Cells were plated onto LB agar plates (containing kanamycin) and incubated at 37° C. overnight.
IV) Colony PCR of NOVABLUE Transformants
To confirm the successful transformation of insert DNA (pET30/insert DNA) in E. coli cells, we selected several colonies of each transformant grown on LB plates (with kanamycin), and performed colony PCR using the same set of Ek/LIC primers as in the amplification of the genes from the Anaplasma genomic DNA. An aliquot of each PCR reaction was analyzed using agarose gel electrophoresis.
For illustration purposes, FIG. 6 shows agarose gel electrophoresis analysis of eight of virB10 transformants in NOVABLUE E. coli. Amplicons of expected size ( ̃1,100 bp) (arrow) were observed following analysis of the PCR reactions. NOVABLUE E. coli colonies containing the pET30/insert DNA were further cultured in LB-kanamycin broth (for the isolation of plasmids).
V) Plasmid Mini-Preps
In order to confirm the presence and sequence accuracy of the cloned insert DNA in the pET30 vector, we performed sequence analysis on the recombinant plasmids. The sequence analysis also provides information that the insert was in-frame of the upstream His-tag sequence. First, we isolated plasmid DNA from the transformed E. coli. WIZARD Plus SV Minipreps DNA Purification system (PROMEGA) was used according to the manufacturer's recommended protocol. The concentration (1 OD260/280=0.5 mg/ml) and the relative purity (OD260/280) of the isolated plasmid DNA preparations were determined by spectrophotometric analysis.
VI) Sequencing Analysis of Insert DNA
We next performed sequence analysis on the isolated plasmid DNA using the APPLIED BIOSYSTEMS 3130 Genetic Analyzer DNA Sequencing instrument. All of the insert DNA were confirmed to be accurate by BLAST analysis and in-frame. As examples, the sequence analysis of the isolated plasmid DNA for virB10 is summarized in FIG. 5. FIG. 5 depicts polynucleotide sequence encoding virB10, together with its deduced amino acid sequence. BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis of the sequences confirmed a match between each of the nucleotide sequences and the published sequences of the respective Anaplasma phagocytophilium genes.
VII) Transformation of BL21 (DE3) E. coli With Recombinant Plasmids
After confirmation of the obtained recombinant plasmids, we proceeded to transform them into BL21 (DE3) competent E. coli (NOVAGEN). Transformation was carried out by removing the appropriate number of 20 μl aliquots of competent cells from −80° C., allowing the tubes to thaw on ice for several minutes, followed by the addition of 1 μl of the plasmid preparation to the cells with gentle stirring. The mixture was incubated on ice for 5 minutes, followed by heating of the tubes for exactly 30 seconds in a 42° C. water bath. The tubes were immediately placed on ice for 2 min. SOC (room temperature) was added, and the reactions were further incubated at 37° C. for 1 hour at 250 rpm. Cells were then plated onto LB agar plated (containing kanamycin) and incubated at 37° C. overnight.
VIII) Colony PCR of BL21 (DE3) Transformants
To confirm the successful transformation of recombinant pET30/insert DNA in BL21 (DE3) E. coli cells, we selected several colonies of each transformant grown on LB plates (with kanamycin), and performed colony PCR using forward and reverse vector-specific primers. An aliquot of each PCR reaction was analyzed using agarose gel electrophoresis. FIG. 7 shows agarose gel electrophoresis analysis of five (5) of virB10 transformants in BL21 (DE3) E. coli. Amplicons of expected size ( ̃1,100 bp) (arrow) were observed following analysis of the PCR reactions. Several BL21 (DE3) E. coli colonies containing the pET30/insert DNA were then processed for recombinant expression.
In addition to virB10, we also confirmed the successful transformation of recombinant pET30/insert DNA for control inserts (i.e., succinate dehydrogenase iron-sulfur and p44).
IX) Expression of Recombinant virB10 Protein in E. coli 
FIG. 8 depicts a flow chart depicting the steps for IPTG induction of recombinant TIVSS proteins in BL21 E. coli. For expression of recombinant TIVSS (rTIVSS) protein virB10 and non-TIVSS proteins (for example, succinate dehydrogenase iron-sulfur submit and p44), BL21 (DE3) E. coli were transformed with the pET30-rTIVSS plasmid DNA containing the respective genes.
The expression was induced with IPTG as follows: 3 ml of LB broth cultures with kanamycin (30 μg/ml final concentration) were inoculated with BL21 transformed with pET30-rTIVSS plasmid. Cultures were grown to mid-log phase (OD600=0.5) at 37° C. with shaking at 250 rpm. When the cultures reached mid-log, the entire 3 ml was added to 100 ml LB broth with kanamycin (30 μg/ml final concentration) and allowed to grow to mid-late log phase (OD600=0.5−1). When the cultures reached mid-late log stage, they were split into two separate 50 ml batches in 250 ml flasks. To one flask, 500 μl of IPTG was added (final concentration of 1 mM). No IPTG was added to the other flask which served as a control for assessing induction. Growth of the IPTG and control cultures was allowed to proceed for 3-3.5 hours at 37° C. with shaking (250 rpm). Cell pellets were then harvested by centrifugation at 3,000 rpm for 15 minutes at 4° C., and subsequently processed with BUGBUSTER Master Mix (NOVAGEN) as described.
X) Isolation and Purification of Recombinant virB10 and P44 Proteins
Isolation of the expressed recombinant virB10 protein was performed using BUGBUSTER Master Mix (NOVAGEN) according to the manufacturer's protocol. After IPTG induction, bacterial cells were harvested from liquid cultures by centrifugation at 3,000 rpm for 15 minutes. Recombinant virB10 protein was isolated both from supernatant and cell pellets. Cell pellets were re-suspended in 5 ml of BUGBUSTER Master Mix (NOVAGEN) by gentle vortexing. The resulting cell suspensions were incubated on a rotating mixer for 20 minutes at room temperature. The mixtures were centrifuged at 4° C. for 20 minutes at 16,000×g to remove the insoluble cellular debris. The supernatant was transferred to a fresh tube for SDS PAGE analysis. The pellet was then processed to isolate the insoluble cytoplasmic fraction, which consists of cell debris and aggregated protein (inclusion bodies). Inclusion body purification was carried out by re-suspending the pellet in the same volume (5 ml) of 1× BUGBUSTER Master Mix used to re-suspend the original cell pellet. The mixtures were vortexed, followed by the addition of 20 ml of 1:10 diluted BUGBUSTER Master Mix. The suspensions were vortexed, and then centrifuged at 5,000×g for 15 minutes at 4° C. to collect the inclusion body fraction. The pellets were re-suspended in 15 ml of 1:10 diluted BUGBUSTER Master Mix, vortexed, and centrifuged at 5,000×g for 15 min. at 4° C. This step was repeated, with the centrifugation carried out for 15 minutes at 16,000×g. The supernatant was discarded, and the pellets re-suspended in 500 μl of PBS. An aliquot of the purified inclusion body fraction was analyzed on an SDS PAGE gel.
(XI) Purification of Recombinant Recombinant virB10 and P44 Proteins Under Urea Denaturing Conditions
The recombinant proteins present within the inclusion body pellets were re-suspended in 4 ml of denaturing lysis/binding buffer. To this mixture was added 1 ml of Ni-NTA His•Bind slurry (Novagen). The suspension was mixed gently on a rotating shaker for 1 hour. The lysate-resin mixture was carefully loaded onto a column placed over a 15 ml conical tube, and the flow-through collected and saved for later analysis. The column was washed with 4 ml of wash buffer collected in another 15 ml conical tube, and the fraction saved for later analysis. The column was washed again with 4 ml of wash buffer, and the fraction saved for later analysis. The recombinant protein was eluted with 5×0.5 ml of elution buffer (pH 5.9) (labeled as E1-E5 in FIG. 10), 5×0.5 ml of elution buffer (pH 5.0) (labeled as E6-E10 in FIG. 10), and 5×0.5 ml of elution buffer (pH 4.9) (labeled as E11-E15 in FIG. 10).
The following buffers were prepared immediately prior to being used:
Lysis Buffer with Urea
100 mM Phosphate buffer
10 mM Tris-Cl
8 M urea
Buffer pH adjusted to 8.0
Wash Buffer with Urea
100 mM Phosphate buffer
10 mM Tris-Cl
8 M urea
Buffer pH adjusted to 6.3
Elution Buffer with Urea (pH 5.9)
100 mM Phosphate buffer
10 mM Tris-Cl
8 M urea
Buffer pH adjusted to 5.9
Elution Buffer with Urea (pH 5.0)
100 mM Phosphate buffer
10 mM Tris-Cl
8 M urea
Buffer pH adjusted to 5.0
Elution Buffer with Urea (pH 4.5)
100 mM Phosphate buffer
10 mM Tris-Cl
8 M urea
Buffer pH adjusted to 4.5
Example 3
IgG/IgM ELISA for Recombinantly Expressed virB10 Protein
We adopted IgG and IgM ELISA assays and evaluated the binding activity of the recombinant proteins towards IgG and IgM. The ELISA procedure involves: (i) coating 96-well micro-titer plates with the recombinant protein at varying concentrations at 4° C. overnight; (ii) adding 5% non-fat milk to block non-specific binding; (iii) adding patients' sera to allow formation of antibody-antigen complex; (iv) detecting the antibody-antigen complex. IFA sero-positive sera served as positive controls, and IFA sero-negative sera served as negative controls. Detection of antibody-antigen complex was performed with the use of horseradish peroxidase.
Patient Study: virB10
IgM ELISA
In these series of studies, we examined recombinant virB10 in an IgM ELISA. Recombinant virB10 protein exhibited a dose-dependent increase in binding towards IgM sero-positive serum (as measured by OD450 nm). IgM ELISA for recombinant virB10 attained a 71.4% sensitivity (FIG. 17) and 90.5% specificity, both of which satisfies the threshold (≧70%) required by industry.
IgG ELISA
Recombinant virB10 protein, when tested in an IgG ELISA, exhibited a dose-dependent increase in binding towards IgG sero-positive serum as measured by OD450 nm. However, the binding levels attained (i.e., 52.4% sensitivity) were below the threshold (≧70%) levels required. IgG ELISA for recombinant virB10 has a specificity of 85.7%, which is within the acceptable range (≧70%) (See, FIG. 17).
ROC Analysis
The raw IgM ELISA data was analyzed with ROC curve determination using MedCalc statistical software. Performance analysis of ROC curve is shown in FIG. 17a. AUC of recombinant virB10 is 0.821 (95% confidence interval; range: 0.672-0.922).
Example 4
Amplification and Cloning of virB10 Protein Fragments
I) PCR Amplification and Ligation into Plasmid Vector
We cloned and recombinantly expressed in E. coli various virB10 protein fragments; namely protein fragments 1-5. Using the antigenicity plot for full-length virB10 (See, FIG. 18), we designed oligonucleotides to amplify 5 (five) fragments encompassing regions of the protein predicted to be antigenic. The location of these fragments relative to that of the full-length virB10 protein is shown in FIG. 12. The nucleotide (SEQ ID No. 12) and amino acid (SEQ ID No. 13) sequences of fragment-1 are shown in FIG. 13. The nucleotide (SEQ ID No. 14) and amino acid (SEQ ID No. 15) sequences of fragment-2 are shown in FIG. 14. The nucleotide (SEQ ID No. 16) and amino acid (SEQ ID No. 17) sequences of fragment-3 are shown in FIG. 15. The nucleotide (SEQ ID No. 18) and amino acid (SEQ ID No. 19) sequences of fragment-4 are shown in FIG. 16. The nucleotide (SEQ ID No. 20) and amino acid (SEQ ID No. 21) sequences of fragment-5 are shown in FIG. 17. Using the cloning strategy detailed in Example 2 (above), we designed and prepared synthetic oligonucleotides ( ̃30 bp in length) and used them in amplifying the various virB10 protein fragments. Table 4 shows the nucleotide sequence of the various oligonucleotides (i.e., SEQ ID Nos. 22-31) used in the PCR amplification reaction.
Using the synthetic oligonucleotides (polynucleotide sequence listed in Table 4) and genomic DNA from Anaplasma phagocytophilum, we successfully amplified five (5) virB10 gene fragments; as well as a non-TIVSS gene (i.e., p44 proteins) (See, FIGS. 19 and 20).
FIG. 19 shows an agarose gel of the amplified virB10 fragments 1-5 prior to processing of the PCR reactions in preparation for ligation into pET30 vector. In preparation for ligation with the vector, the PCR amplification reactions were treated to remove any remaining nucleotides, primers, and reaction components. The resulting PCR products were then treated with T4 DNA polymerase and ligated into pET30 using standard protocols. Ligation of the virB10 fragment insert DNA (including succinate dehydrogenase iron-sulfur and p44 protein insert DNAs) was performed as described below.
II) T4 Polymerase Treatment of PCR Products and Ligation into pET30 Vector
In order to ligate the cloned fragment insert DNAs with the plasmid vector, it is necessary to create compatible ends between the amplicon and the chosen vector (e.g., pET30 Ek/LIC). We generated overhangs compatible with the Ek/LIC cloning vector on the insert DNA by T4 DNA polymerase treatment of the PCR amplicon. We ligated the treated amplicons into the expression vector to form pET30/insert DNA. FIG. 22 depicts the pET30 vector containing the insert DNA (Fragments 1-5).
III) Transformation of Recombinant Clones into NOVABLUE E. coli 
In these series of experiments, we transformed the ligated DNAs (annealing reaction) into host bacterial cells (NOVABLUE E. coli). The ligated DNAs were virB10 fragments 1-5 amplicons. We chose NOVABLUE E. coli because this bacterial strain is optimized for producing a stable cell line containing a recombinant insert (see, NOVABLUE Ek/LIC manual). Transformation into NOVABLUE competent E. coli (NOVAGEN) was performed using standard protocols. First, appropriate numbers of 20 μl aliquots of competent cells were prepared from −80° C., and allowed to thaw on ice for several minutes, followed by the addition of 1 μl of the annealing reaction and gentle stirring. The mixture was further incubated on ice for an additional 5 minutes, followed by heating the tubes for 30 seconds in a 42° C. water bath. The tubes were immediately placed on ice for 2 minutes. SOC (Super Optimal broth with Catabolite repression medium, containing 2% w/v bacto-tryptone, 0.5% w/v bacto-yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM glucose) (at room temperature) was added into the tubes, and the reactions were further incubated for 1 hour at 37° C. with shaking (250 rpm). Cells were plated onto LB agar plates (containing kanamycin) and incubated at 37° C. overnight.
IV) Colony PCR of NOVABLUE Transformants
To confirm the successful transformation of insert DNA (pET30/insert DNA) in E. coli cells, we selected several colonies of each transformant grown on LB plates (with kanamycin), and performed colony PCR using the same set of Ek/LIC primers as in the amplification of the genes from the Anaplasma genomic DNA. An aliquot of each PCR reaction was analyzed using agarose gel electrophoresis.
FIG. 20 shows agarose gel electrophoresis analysis of three virB10 transformants for each fragment (1, 2, 3, and 5) in NOVABLUE E. coli. FIG. 21 shows agarose gel electrophoresis analysis of six virB10 transformants for fragment 4 (arrows). NOVABLUE E. coli colonies containing the pET30/insert DNA were further cultured in LB-kanamycin broth (for the isolation of plasmids).
V) Plasmid Mini-Preps
In order to confirm the presence and sequence accuracy of the cloned insert DNA in the pET30 vector, we performed sequence analysis on the recombinant plasmids. The sequence analysis also provides information that the insert was in-frame of the upstream His-tag sequence. First, we isolated plasmid DNA from the transformed E. coli. WIZARD Plus SV Minipreps DNA Purification system (PROMEGA) was used according to the manufacturer's recommended protocol. The concentration (1 OD260/280=0.5 mg/ml) and the relative purity (OD260/280) of the isolated plasmid DNA preparations were determined by spectrophotometric analysis.
VI) Sequencing Analysis of Insert DNA
We next performed sequence analysis on the isolated plasmid DNA using the APPLIED BIOSYSTEMS 3130 Genetic Analyzer DNA Sequencing instrument. All of the insert DNA were confirmed to be accurate by BLAST analysis and in-frame. BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis of the sequences confirmed a match between the nucleotide and deduced amino acid sequences for each of the fragments and the published sequences of the Anaplasma phagocytophilum virB10 gene.
We next performed sequence analysis on the isolated plasmid DNA using the APPLIED BIOSYSTEMS 3130 Genetic Analyzer DNA Sequencing instrument. All of the insert DNA were confirmed to be accurate by BLAST analysis and in-frame. BLAST (Basic Local Alignment Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis of the sequences confirmed a match between the nucleotide and deduced amino acid sequences for each of the fragments and the published sequences of the Anaplasma phagocytophilum virB10 gene.
VII) Transformation of BL21 (DE3) E. coli With Recombinant Plasmids
After confirmation of the obtained recombinant plasmids, we proceeded to transform them into BL21 (DE3) competent E. coli (NOVAGEN). Transformation was carried out by removing the appropriate number of 20 μl aliquots of competent cells from −80° C., allowing the tubes to thaw on ice for several minutes, followed by the addition of 1 μl of the plasmid preparation to the cells with gentle stirring. The mixture was incubated on ice for 5 minutes, followed by heating of the tubes for exactly 30 seconds in a 42° C. water bath. The tubes were immediately placed on ice for 2 min. SOC (room temperature) was added, and the reactions were further incubated at 37° C. for 1 hour at 250 rpm. Cells were then plated onto LB agar plated (containing kanamycin) and incubated at 37° C. overnight.
VIII) Colony PCR of BL21 (DE3) Transformants
To confirm the successful transformation of recombinant pET30/insert DNA in BL21 (DE3) E. coli cells, we selected several colonies of each transformant grown on LB plates (with kanamycin), and performed colony PCR using forward and reverse vector-specific primers. An aliquot of each PCR reaction was analyzed using agarose gel electrophoresis. Amplicons of expected size for each fragment were observed following analysis of the PCR reactions (data not shown).
Example 5
Expression and Purification of virB10 Protein Fragments
I) Expression of Recombinant virB10 Fragments 1-5 in E. coli 
In order to express fragments 1-5 of virB10, the Overnight ExpressTM Autoinduction System 1 (NOVAGEN) was used. In each 500 ml flask (one baffled and one flat bottom per fragment), 110 ml of LB broth was added. From the Autoinduction kit, 0.02 volume of OnExTM Solution 1, 0.05 volume of OnEx Solution 2, and 0.001 volume of OnEx Solution 3 were added to 1 volume LB medium (glucose free). Kanamyacin was added to a final concentration of 30 μg/ml. LB medium was inoculated with isolated colonies from the plates, and incubated overnight (approximately 16 hours) at 37° C. with shaking at 250 rpm.
The following day, each culture of the fragments was spun down for 10 minutes at 10,000×g. The supernatant was decanted, and 15 ml of BUGBUSTER Master Mix (NOVAGEN) was used to resuspend each pellet thoroughly. The cell suspension was incubated in room temperature on a shaker at slow speed for 20 minutes, and was then centrifuged at 4° C. at 16,000×g for 20 minutes to separate the soluble cytoplasmic fraction (supernatant) from the insoluble cytoplasmic fraction (pellet). The pellets were resuspended in 15 ml BUGBUSTER, after which 6 volumes of 1:10 diluted BUGBUSTER was added to each and then vortexed for 1 minute. The resuspension was centrifuged at 4° C. at 5,000×g for 15 minutes, and the supernatant was saved as an insoluble wash. The pellet was resuspended in half the original culture volume of 1:10 diluted BUGBUSTER, mixed by vortexing, and centrifuged at 4° C. at 5,000×g for 15 minutes. This step was repeated twice, with the final spin at 16,000×g. The pellets (inclusion bodies) were then kept at −70° C. until needed for further purification.
The soluble cytoplasmic fractions and the insoluble washes were analysed on SDS-PAGE gels, which showed that fragments 1 and 2 were found in the soluble fractions (FIG. 23), and fragments 3-5 were present in the insoluble (inclusion body) fractions (FIGS. 26, 27, 30). A COOMASSIE-stained gel and Western blot detection of fragments 1 and 2 using an antibody directed against the 6× His-tag shows that a these recombinant proteins were present in the soluble fraction (FIG. 24). A COOMASSIE-stained gel and Western blot detection of fragments 3, 4, and 5 using an antibody directed against the 6× His-tag shows that a majority of these recombinant proteins was present in the insoluble (inclusion body) fraction (FIGS. 29 and 32).
For purification of fragments 1 and 2 from the soluble fraction, Ni-NTA Buffer Kit (NOVAGEN) and Ni-NTA His•Bind Resin (NOVAGEN) were used. In order to equilibrate the resin, 30 ml 1× Binding Buffer (equal to the amount of the soluble fraction) was added to 5 ml resin, and the mixture was incubated on a shaker in 4° C. for 10 min, prior to the tubes being placed in an upright position at room temperature to facilitate the settling of the resin at the bottom of the tubes. 30 ml of the Binding Buffer from the top was taken out and replaced with the soluble fraction. The resin/soluble fraction mixture was then incubated on a shaker at 4° C. for 1 hour. The mixture was then decanted into an empty column. Using a slow drip, the flow-through was collected. Taking careful steps to avoid allowing the resin to become dry at any time, 4 ml or 1× Wash buffer was added twice. Lastly, 5×0.5 ml of 1× Elution Buffer was added to the resin to collect the protein. The flow-through, wash buffers and elution buffers were analyzed on an SDS-PAGE gel to confirm the successful purification of the proteins, and to determine in which fractions the proteins were eluted. SDS PAGE analysis confirmed that a majority of recombinant fragment-1 and 2 eluted from the column in elution fractions 1-3 (FIG. 25).
II) Purification of Recombinant virB10 Fragments 3, 4, and 5 Under Urea Denaturing Conditions
The inclusion body fractions containing recombinant fragments 3-5 were purified under urea denaturing conditions as previously described for full-length virB10 and p44 proteins using freshly prepared buffers containing urea. Nickel column purification of fragments 3 and 4 is shown in FIG. 28.
Example 6
IgG/IgM ELISA for Recombinantly Expressed virB10 Protein Fragments
We performed IgG and IgM ELISA assays and evaluated the binding activity of the recombinant virB10 protein fragments towards IgM and/or IgG.
Patient Study: virB10 Protein Fragments 1-5
virB10 Protein Fragments 1 and 2
In these studies, we examined recombinant virB10 fragments 1 and 2 in IgG ELISAs. Fragment 1 exhibited a dose-dependent increase in binding towards IgG sero-positive serum (as measured by OD450 nm). IgG ELISA for recombinant fragment 1 attained a 76.2% sensitivity (FIG. 33) and 71.4% specificity, both of which satisfies the threshold (≧70%) required by industry.
Fragment 2 exhibited a dose-dependent increase in binding towards IgG sero-positive serum (as measured by OD450 nm). IgG ELISA for recombinant fragment 1 attained a 81.0% sensitivity (FIG. 33). However, the specificity attained of 57.1%.
Recombinant fragment 1, when tested in an IgM ELISA, exhibited a dose-dependent increase in binding towards IgM sero-positive serum as measured by OD450 nm. IgM ELISA for recombinant fragment 1 attained a 85.6% sensitivity (FIG. 34) and 85.6% specificity.
Recombinant fragment 2 when tested in an IgM ELISA, exhibited a dose-dependent increase in binding towards IgM sero-positive serum as measured by OD450 nm. IgM ELISA for recombinant fragment 1 attained a 84.6% sensitivity (FIG. 34) and 93.9% specificity.
Combined virB10 Protein Fragments 1 and 2
In the next series of experiments, we sought to test the usefulness of combining fragments 1 and 2. Recombinant fragments 1 and 2, when combined and used for ELISA analysis, exhibited a dose-dependent increase in binding towards IgM sero-positive serum as measured by OD450 nm. As shown in FIG. 35, the combination of fragments 1 and 2 attained an 81.0% sensitivity and 85.7% specificity.
Recombinant fragments 1 and 2, when combined and used for ELISA analysis, exhibited a dose-dependent increase in binding towards IgG sero-positive serum as measured by OD450 nm. As shown in FIG. 36, the use of a combination of fragments 1 and 2 attained ELISA with 76.% sensitivity and 76.2% specificity.
virB10 Protein Fragments 3 and 4
In the next series of studies, we examined recombinant virB10 fragments 3 and 4 in IgG ELISAs. Fragment 3 exhibited a dose-dependent increase in binding towards IgG sero-positive serum (as measured by OD450 nm). IgG ELISA for recombinant fragment 3 attained a 85.7% sensitivity (FIG. 37) and a specificity of 61.9%.
Fragment 4 exhibited a dose-dependent increase in binding towards IgG sero-positive serum (as measured by OD450 nm). IgG ELISA for recombinant fragment 4 attained a 81.0% sensitivity (FIG. 37) and the specificity of 61.9%.
virB10 Protein Fragment 5
In the final series of studies, we examined recombinant virB10 fragments 5 in IgG ELISAs. Fragment 5 exhibited a dose-dependent increase in binding towards IgG sero-positive serum (as measured by OD450 nm). IgG ELISA for recombinant fragment 3 attained 76.2% sensitivity (FIG. 38) and a specificity of 66.7%.
Experimental Protocol
Anaplasma IgG ELISA

    1. Antigen coating concentration 0.5 μg/ml in carbonate buffer (pH 9.6) (100 μl per well). Coating overnight in 4° C.
    2. Wash three time in PBST buffer (0.5% TWEEN-20)
    3. Block with 200 μl blocker buffer (casein in PBS, Thermo Sci. #37528). Incubate for 1 hour in room temperature
    4. Wash three times with PBST buffer (0.5% TWEEN-20)
    5. Add 100 μl 1:200 diluted human sera (dilution buffer: 1:20 casein buffer in PBST). Incubate for 1 hour in room temperature
    6. Wash four times with PBST buffer (0.5% TWEEN-20)
    7. Add goat anti-human IgG antibody (1:15,000 diluted in casein dilution buffer (1:20 casein buffer in PBST). Incubate for 1 hour in room temperature
    8. Wash four times with PBST buffer (0.5% Tween-20)
    9. Add 100 μTBM substrate. Incubate in room temperature for 3 minutes
    10. Stop the reaction with 2N HCl
    11. Read the result at OD450 


Anaplasma IgM ELISA

    1. Antigen coating concentration 0.125 μg/ml in carbonate buffer (pH 9.6) (100 μl per well). Coating overnight in 4° C.
    2. Wash three time in PBST buffer (0.5% TWEEN-20)
    3. Block with 200 μl blocker buffer (casein in PBS, Thermo Sci. #37528). Incubate for 1 hour in room temperature
    4. Wash three times with PBST buffer (0.5% TWEEN-20)
    5. Dilute human sera in GullSorbTM (1:10) to prepare mixture 1. Incubate in room temperature for 5 minutes. Dilute incubated mixture 1 in sample dilution buffer (1:20 casein buffer in PBST). Therefore, the total dilution factor for human sera is 1:100
    6. Add 100 μl 1:100 diluted human sera to the plate. Incubate for 1 hour in room temperature
    7. Wash four times with PBST buffer (0.5% TWEEN-20)
    8. Add goat anti-human IgM antibody (1:10,000 diluted in casein dilution buffer (1:20 casein buffer in PBST). Incubate for 1 hour in room temperature
    9. Wash four times with PBST buffer (0.5% TWEEN-20)
    10. Add 100 μl TBM substrate. Incubate in room temperature for 3 minutes
    11. Stop the reaction with 2 N HCl
    12. Read the result at OD450 


All publications and patents cited in this specification are herein incorporated by reference in their entirety. Various modifications and variations of the described composition, method, and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments and certain working examples, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Various modifications of the above-described modes for carrying out the invention that are obvious to those skilled in the filed of molecular biology, recombinant expression and related fields are intended to be within the scope of the following claims.







TABLE 1







Oligonucleotide Sequences Used in


Gene Amplification for Anaplasma


phagocytophilum Encoding virB10


and Non-TIVSS Protein Components










Recombinant
NCBI

Gene


TIVSS & Non-
Accession

Amplifi-


TIVSS Protein
#
Oligonucleotides
cation





virB10
YP_505896
Fwd: 5′-gacgacga
Yes




caagatggctgacgaa





ataaggggttc-3′





(SEQ. ID No. 1)





Rev: 5′-gaggagaa





gcccggtctacctcac





cgcatcacg-3′





(SEQ. ID No. 2)



 


Succinate
YP_504786
Fwd: 5′-gacgacga
Yes


Dehydrogenase,

caagatggtgcagttt



iron-sulfur

tctttgcc-3′



subunit

(SEQ. ID No. 3)





Rev: 5′-gaggagaa





gcccggtctagagctc





caatccttttatc-3′





(SEQ. ID No. 4)



 


p44-8
YP_504769
Fwd: 5′-gacgacga
Yes


Outer Membrane

caagatgctaaggctc



Protein

atggtgatgg -3′





(SEQ ID No: 5)





Rev: 5′-gaggagaa





gcccggttcaaaaacg





tattgtgcgacg-3′





(SEQ ID No: 6)

















TABLE 2







Recombinant Expression of virB10 and Non-TIVSS


Proteins in Anaplasma phagocytophilum









Recombinant TIVSS and

Recombinant


Non-TIVSS Protein
NCBI Accession Nos.
Expression





virB10
YP_505896
Yes



(SEQ ID No. 7)


Succinate Dehydrogenase,
YP_504786
No


iron-sulfur subunit
(SEQ ID No. 8)


P44-8 Outer Membrane
YP_504769
Yes


Protein
(SEQ ID No. 9)
















TABLE 3







IgM/IgG ELISA Assay for Recombinant virB10 and p44









Recombinant




TIVSS and


Non-TIVSS Proteins
IgM ELISA
IgG ELISA





virB10
Sensitivity = 71.4%
Sensitivity = 57.1%



Specificity = 85.7%
Specificity = 76.2%


p44 Outer
Sensitivity = 81%
Sensitivity = 42%-71.4%


Membrane Protein
Specificity = 90.5%
Specificity = 71.4%-100%
















TABLE 4







Primers for Generation of Polynucleotides Encoding


Five (5) Recombinant Protein Fragments


of TIVSS virB10 in Anaplasma phagocytophilum









TIVSS virB10




Fragments
Primers
Nucleotide Sequences





Fragment 1
Forward
5′-gacgacgacaagatgatggctgac




gaaataag-3′ SEQ ID NO: 22



Reverse
5′-gaggagaagcccggttatggcgtc




aagattct-3′ SEQ ID NO: 23


 


Fragment 2
Forward
5′-gacgacgacaagatgcagattcct




cgtgttat-3′ SEQ ID NO: 24



Reverse
5′-gaggagaagcccggttattccttc




ccgccaac-3′ SEQ ID NO: 25


 


Fragment 3
Forward
5′-gacgacgacaagatggaagatgct




cggtttac-3′ SEQ ID NO: 26



Reverse
5′-gaggagaagcccggttagcccact




ttattatc-3′ SEQ ID NO: 27


 


Fragment 4
Forward
5′-gacgacgacaagatgttacctcat




ggcgttga-3′ SEQ ID NO: 28



Reverse
5′-gaggagaagcccggttactacctc




accgcatc-3′ SEQ ID NO: 29


 


Fragment 5
Forward
5′-gacgacgacaagatgccgcttgta




atgcctac-3′ SEQ ID NO: 30



Reverse
5′ gaggagaagcccggttataaaatg




accctgga-3′ SEQ ID NO: 31
















TABLE 5







ELISA Sensitivity and Specificity for


Various virB10 Protein Fragments











Recombinant





TIVSS virB10



Fragments
IgG ELISA
IgM ELISA







Fragment 1
Sensitivity = 76.2%
Sensitivity = 85.7%




Specificity = 71.4%
Specificity = 85.7%



Fragment 2
Sensitivity = 81.0%
Sensitivity = 84.6%




Specificity = 57.1%
Specificity = 93.9%



Fragments 1 + 2
Sensitivity = 76.2%
Sensitivity = 81.0%




Specificity = 76.2%
Specificity = 85.7%



Fragment 3
Sensitivity = 85.7%
Not determined




Specificity = 61.9%



Fragment 4
Sensitivity = 81.0%
Not determined




Specificity = 61.9%



Fragment 5
Sensitivity = 76.2%
Not determined




Specificity = 66.7%

## Claims

1. A method of detecting the presence of an antibody against Anaplasma phagocytophilum in a biological sample of a mammal comprising:
(i) immobilizing an isolated polypeptide antigen onto a surface, wherein said immobilized isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15;
(ii) contacting said immobilized isolated polypeptide antigen with the biological sample of the mammal, under conditions that allow formation of an antibody-antigen complex, said biological sample containing an antibody against Anaplasma phagocytophilum, wherein said antibody binds to said immobilized isolated polypeptide antigen and forms a complex of the antibody and the polypeptide antigen; and
(iii) detecting the formation of said antibody-polypeptide antigen complex, wherein said detected antibody-polypeptide antigen complex is indicative of the presence of said antibody against Anaplasma phagocytophilum in said biological sample.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said antibody is an IgG or IgM.

4. The method of claim 1, wherein said method is an ELISA.

5. The method of claim 4, wherein said ELISA has a sensitivity of at least >70%.

6. The method of claim 4, wherein said ELISA has a specificity of at least >70%.

7. A method of diagnosing an infection of Anaplasma phagocytophilum in a mammal by detecting the presence of an antibody against said Anaplasma phagocytophilum comprising the steps of:
(i) obtaining a biological sample from the mammal, wherein said mammal is suspected of having the Anaplasma phagocytophilum infection;
(ii) immobilizing an isolated polypeptide antigen onto a surface, wherein said immobilized isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 15;
(iii) contacting said immobilized isolated polypeptide antigen with said biological sample, under conditions that allow formation of an antibody-antigen complex, said biological sample containing an antibody against Anaplasma phagocytophilum, and wherein said antibody binds to said immobilized isolated polypeptide antigen and forms a complex of the antibody and the polypeptide antigen; and
(iv) detecting said antibody-polypeptide antigen complex, wherein said detected antibody-polypeptide antigen complex is indicative of the presence of said antibody against Anaplasma phagocytophilum in said biological sample.

8. The method of claim 7, wherein the isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 13.

9. The method of claim 7, wherein the isolated polypeptide antigen has the amino acid sequence set forth in SEQ ID NO: 15.

10. The method of claim 7, wherein the mammal is a human.

11. The method of claim 7, wherein said biological sample is whole blood.

12. The method of claim 7, wherein the antibody is IgG or IgM.

13. The method of claim 7, wherein said contacting step is performed at room temperature for about 1 hour.