MXPA01005378A - Homologous 28-kilodalton immunodominant protein genes of ehrlichia canis - Google Patents

Abstract

The present invention is directed to the cloning, sequencing and expression of homologous immnunoreactive 28-kDa protein genes, ECa28-1 and ECa28SA3, from a polymorphic multiple gene family of Ehrlichia canis. A complete sequence of another 28-kDa protein gene, ECaSA2, is also provided. Further disclosed is a multigene locus encoding all five homologous 28-kDa protein genes of Ehrlichia canis. Recombinant Ehrlichia canis 28-kDa proteins react with convalescent phase antiserum from an E. canis-infected dog.

Description

GENES OF THE IMMUNODOMINANT PROTEIN OF 28-KILODALTOMS, HOMOLOGA, OF EHRLICHIA CANIS AND USE OF THEM BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to the molecular cloning and characterization of the 28-kDa homologous protein genes in Ehrli chla canis and a multi-gene site encoding the 28-kDa homologous proteins of Ehrlichia cani s and uses of them. Description of Related Art Canine ehrlichiosis, also known as canine tropical pancytopenia, is a condition caused by rickettsia that carry ticks in dogs, first described in Africa in 1935 and in the United States in 1963 (Donatien and Lestoquard, 1935 Ewing, 1963). The condition is best recognized after an epizootic epidemic has occurred in United States military dogs during the Vietnam War (Walker et al., 1970). The etiological agent of canine ehrlichiosis is Ehrlichia cani s, a small intracellular bacterium.

REF: 129792 gram-negative parasite that exhibits tropism for mononuclear phagocytes (Nyindo et al., 1971) and is transmitted by the dog brown tick, Rhipi c pha l us sanguineus (Groves et al., 1975). The progression of canine ehrlichiosis occurs in three phases; acute, subclinical and chronic. The acute phase is characterized by the presence of fever, anorexia, depression, lymphadenopathy, and mild thrombocytopenia (Troy and Forrester, 1990). Typically, dogs recover from the acute phase, but they become carriers of the organism persistently infected without clinical symptoms of the disease for months or even years (Harrus et al., 1998). A chronic phase develops in some cases, which is characterized by thrombocytopenia, hyperglobulinemia, anorexia, emaciation, and hemorrhage, particularly epistaxis, followed by death (Troy and Forrester, 1990). The taxonomic analysis based on the 16S rRNA gene has determined that E. Canis and E. Chaffeensis, the etiological agent of human monocytic ehrlichiosis (HME), are closely related (Anderson et al., 1991, Anderson et al., Dawson et al., 1991; Chen et al., 1994). A considerable cross-reactivity of the 64, 47, 40, 30, 29 and 23 kDa antigens has been reported between E. Cani \ s and E. homologs according to the analysis by peripheral blood PCR of 5 days after the immunogenic test (Ohashi et al., 1998). Molecular cloning of similar but not identical 28-kDa genes placed in tandem of E. canis homologous to the E. chaffensis omp-l gene family and the C. rumanin ti um map-1 gene (Reddy et al. al., 1998). The previous technique is deficient due to the absence of cloning and characterization of new genes of the immunoreactive 28-kDa homologous protein of Ehrli chia canis and a single multi-gene site containing the genes of the homologous 28-kDa protein. In addition, the prior art is deficient due to the absence or lack of recombinant proteins of such Ehrlichia canis immunoreactive genes. The present invention fulfills this need for a long time and desire in the art.

BRIEF DESCRIPTION OF THE INVENTION The present invention describes the molecular cloning, sequencing, characterization, and expression of the genes of the mature immunoreactive 28-kDa protein homologous of Ehrlí chia canis (termed Eca28-1, ECa28SA3 and ECa28SA2), and the identification of a single place (5.592-kb) ^ ygfcs | ? gj containing five 28-kDa protein genes from Ehrli chia canis (ECa28SAl, ECa28SA2, ECa28SA3, Eca28-1 and ECa28-2). The comparison of E. Chaffeensis between the genes of the 28-kDa protein of E. Canis revealed that ECa28-l shares the highest amino acid homology with the multiple gene family of E. Chaffensis omp-l and is highly conserved between E. Canis isolated. It was predicted that the five 28-kDa proteins have signal peptides that result in mature proteins, and have amino acid homology in the range of 51 to 72%. The analysis of intergenic regions revealed hypothetical promoter regions for each gene, suggesting that these genes can be expressed independently and differentially. The non-coding intergenic regions vary in size from 299 to 355-bp, and are from 48 to 71 homologs. In one embodiment of the present invention, DNA sequences encoding an immunoreactive 30-kDa protein from Ehrlichia canis are provided. Preferably, the protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO. 4 and SEQ ID No. 6, and the gene has a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5 and is a member of a family of multiple polymorphic genes. Generally, the protein has an N-terminal signal sequence that was cleaved after the post-translational process resulting in the production of a mature 28-kDa protein. Still, preferably the DNAs encoding the 28-kDa proteins are contained in a single multi-gene site, which have the size of 5,592 kb and which encode all five proteins of 28-kDa homologous Ehrlichia canis. In another embodiment of the present invention, there is provided an expression vector comprising a gene encoding a 28-kDa immunoreactive protein of Ehrlichia canis, capable of expressing the gene when the vector is introduced into the cell. In yet another embodiment of the present invention, there is provided a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6. Preferably, the sequence of amino acid is encoded by the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. Preferably, the recombinant protein comprises four variable regions which are exposed in the MtefctMÜM-Éa ^ BaMaA? Atfkri? surface, hydrophilic and antigenic. The recombinant protein can be useful as an antigen. In yet another embodiment of the present invention, there is provided a method for producing the recombinant protein, comprising the steps of obtaining a vector comprising an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID No. 4 and SEQ ID No. 6 operatively linked to a promoter; transfecting the vector in a cell; and culturing the cell under conditions effective for the expression of the expression region. The invention can also be described in certain embodiments as a method of inhibiting the infection of Ehrli chía canis in a subject, which includes the steps of: identifying a subject suspected of being exposed or infected with Ehrli chia canis; and administering a composition comprising a 28-kDa antigen of Ehrlichia canis in i i an amount effective to inhibit an infection of Ehrlichia canis. Inhibition can occur through any means such as, for example, the stimulation of subjects' mood or cellular immune responses, normal antigen of 28-kDa, or even compete with the antigen for interaction with some agents in the body of the subjects. Other aspects, features and additional advantages of the present invention will be apparent from the following description of the present preferred embodiments of the invention given for the purpose of the description. BRIEF DESCRIPTION OF THE DRAWINGS 10 So that the subject in which the characteristics, advantages and aforementioned objects of the invention, as well as others that are clear, will be reached and can be understood in detail; furthermore the particular descriptions of the invention briefly summarized above can be made by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings are part of the specification. It will be noted, however, that the appended drawings illustrate the preferred embodiments of the invention 20 and are therefore not considered to limit its scope. Figure 1 shows the nucleic acid sequence (SEQ ID NO: 1) and the deduced amino acid sequence (SEQ ID No.2) of the Eca28-1 gene which includes the * «Fc -» »~ *. ~ *. * m * «??? t * ~« *, ^ É ^ encoding 5 'and 3' adjacent. The ATG start codon and the TAA termination are shown in bold, and the leader signal sequence of 23 amino acids is underlined. Figure 2 shows the SDS-PAGE of the expressed 50-kDa recombinant Eca28-l-thioredoxin fusion protein (lane 1), arrow) and the 16-kDa thioredoxin control (lane 2, arrow), and the corresponding immunoblotting of the recombinant Eca28-l-thioredoxin fusion protein recognized by the canine antiserum of the convalescent E. canis phase (lane 3). ). Thyroredoxin control was not detected by E. canis antiserum (not shown). Figure 3 shows the alignment of the amino acid sequences of the protein Eca28-1 (SEQ ID NO: 2), and ECa28SA2 (partial sequence, SEQ ID NO: 7) and ECa28SAl (SEQ ID NO: 8), E. chaffeensi s P28 (SEQ ID NO.9), family E. chaffensis OMP-1 (SEQ ID Nos: 10-14) and C. Ruminantiu MAP-1 (SEQ ID No. 15). The amino acid sequence ECa28-l is presented as the consensus sequence. The amino acids not shown are identical for Eca28-1 and are represented by a dot. Divergent amino acids are shown with the corresponding abbreviation of a letter. The intervals entered for the maximum alignment of the. Amino acid sequences are denoted by a hyphen. The jjgjj'J'jj ^ variable regions are underlined and denoted (VR1, VR2, VR3, and VR4). The arrows indicate the cleavage site of the signal peptidase predicted by the signal peptide. Figure 4 shows the phylogenetic relationship of E. canis Eca28-1 with Eca28SA2 (partial sequence) and Eca28SAl, the 6 members of the multiple gene family of E. chaffeensis omp-l, and the amino acid sequences deduced from C. Rumaninti um map-1 using the construction of an unbalanced tree. The length of each pair of each branch represents the distance between the amino acid sequence of the pairs. The scale measures the distance between the sequences. Figure 5 shows Southern blot analysis of E. cani s genomic DNA completely digested with the six individual restriction enzymes and hybridized with a probe labeled with DIG Eca28-1 (lanes 2-7); the molecular weight markers labeled with DIG (lanes 1 and 8). Figure 6 shows the comparison of characteristics of the predicted protein of ECa28-l (Jake strains) and E. chaffeensis P28 (Arkansas strains). The surface probability predicts surface residues using a hexapeptide window. A surface residue is ^^^^ j ^ j ^ any residue with an accessible surface area of water > 2.0 n mz. A hexapeptide with a value higher than 1 is considered as a surface region. The antigenic index predicts potential antigenic determinants. Regions with a value above zero are potential antigenic determinants. Portions of T cells localize the antigenic determinants of potential T cells using a 5 amino acid portion with a 1-glycine or polar residue, 2-hydrophobic residue, residue 3-h? Drofóbico, 4-hydrophobic residue or proline, and 5- polar residue or glycine. The scale indicates the positions of the amino acids. Figure 7 shows the nucleic acid sequences and amino acid sequences deduced from the genes Eca28SA2 of the E canis protein of 28-kDa (nucleotide 1-849: SEQ ID No. 3, amino acid sequence: SEQ ID No. 4) and ECa28SA2 (nucleotide 1195-2031: SEQ ID NO: 5) : SEQ ID No. 6) including non-coding intergenic sequences (NC2, nucleotide 850-1194: SEC ID No. 31). The ATG start codon and the stop codons are shown in black. I Figure 8 shows schematically the place of the gene of the five E canis proteins of 28-kDa (5,592-Kb) indicating the genomic orientation and the non-coding intergenic regions (28NC1-4). The 28-kDa protein genes showing sites 1 and 2 (shading) have been described (McBride et al., 1999, Reddy et al., 1998, Ohashi et al., 1998). The complete sequence of ECaSA2 and a new designated 28-kDa protein gene (ECa28SA3- not shaded) were sequenced. The non-coding intergenic regions (28NC2-3) between ECaSA2, ECa28SA3 and ECa28-l were supplemented by joining the previously unbound site 1 and 2. Figure 9 shows the phylogenetic relationship of the five members of the E canis protein of 28- kDa based on amino acid sequences that use the construction of an unbalanced tree. The length of each pair of branches represents the distance between the pairs of amino acids. The scale measures the distance between the sequences. Figure 10 shows the alignment of the intergenic non-coding nucleic acid sequences of the gene of the 28-kDa protein of E. canis (SEQ ID No. 30-33). The nucleic acids not shown, denoted by a dot (.), Are identical for the noncoding region 1 (28NC1). The divergence is shown with the corresponding abbreviation of a letter. The intervals - '* * - *' "- *** • '- - * - introduced for the maximum alignment of the amino acid sequences are denoted by a dash (-). The putative transcriptional promoter regions (-10 and -35) and the ribosome-binding site (RBS) are enclosed in a box.

DETAILED DESCRIPTION OF THE INVENTION The present invention describes the cloning, Sequencing and expression of homologous genes that encode a 30-kilodalton (kDa) protein of Ehrli chia canis. We also performed a comparative molecular analysis of homologous genes among seven isolated E. canis and the multiple gene family E. chaffeensis omp-l. Two new genes of the 28-kDa protein were identified, ECa28-l and ECa28SA3. The ECa28-l has an open reading structure of 834-bp that encodes a protein of 278 amino acids (SEQ ID No. 2) with a predicted molecular mass of 30.5-kDa. An N-terminal signal sequence was identified suggesting that the protein is post-translationally modified to a mature 27.7-kDa protein. The ECa28SA3 has an open reading structure of 840-bp that encodes a protein of 280 amino acids (SEQ ID No. 6). I encodes a 28-kDa immunoreactive protein from Ehrl i chia canis and that is able to express the gene when the veator is enter the cell. Still, in another embodiment of the present invention, a recombinant protein comprising a amino acid sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 6. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group consisting of consists of SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5. Preferably, the recombinant protein comprises four variable regions that are exposed on the hydrophilic and antigenic surface. Even preferably, the recombinant protein is an antigen. In still another embodiment of the present invention, there is provided a method for producing the recombinant protein, comprising the steps of obtaining a vector comprising an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No. 6 operatively linked to a promoter; transfect the vector to a cell; and cultivate cell under effective conditions for the expression of region of expression. The invention can also be described in certain embodiments as a method of inhibiting the infection of Ehrlichia canis in a subject, comprising the steps of: identifying a subject suspected of being exposed or infected with Ehrlichia canis; and administer a composition comprising a 28-kDa antigen of Ehrlichia canis in an effective amount to inhibit an infection by Ehrl i chia canis. Inhibition can occur through any means such as for example; the stimulation of a person's mood or cellular immune responses, or by other means such as the inhibition of the normal function of the 28-kDa antigen, or even by competition with the antigen by interaction with some agent in the subject's body In accordance with the present invention, conventional molecular biology, I microbiology, and recombinant DNA techniques can be employed within the state of the art. Such techniques are explained II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis"! (M.J. I Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription and Translation" [B.D. Hames & S.J. Higgins eds. (1984)]; "Animal Cell Culture "[RI Freshney, ed (1986)]," Immobilized Cells And Enzymes "[IRL Press, (1986)], B. Perbal," A Practical Guide To Molecular Cloning "(1984) .Therefore, if they appear here the following terms will have the definitions set forth above. A "replicon" is any genetic element (eg, plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; that is, capable of replication under its own control. A "vector" is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment can adhere in order to bring about replication of the attached segment. i "A DNA molecule" refers to the polymeric form of the deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in their form of either single-stranded or double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any of the particular tertiary forms. Thus, this term includes the double-stranded DNA found, inter. Alia, in DNA molecules (for example, restriction fragments), viruses, plasmids, and chromosomes. In the discussion of the structure of the present, according to the normal convention of providing only the sequence in the 3 'to 5' direction along the non-transcribed DNA strand (ie, the strand has a homologous sequence). for mRNA). A "sequence that encodes" DNA is a sequence the 3 'direction for the coding sequence.

The translational and i i transcriptional control sequences are DNA regulatory sequences, i such as promoters, enhancers, polyadenylation signals, and terminators, and the like, which provide for the expression of i I a sequence encoding a host cell. A "promoter sequence" is a DNA regulatory region capable of binding the RNA polymerase in a cell and initiating the transcription of a sequence encoding the i direction 3 '. For purposes of defining the present invention, the promoter sequence is linked to its 3 'terminus via the transcription initiation site and extends in the 5' direction to include the minimum number of bases or elements necessary to initiate transcription at detectable levels mentioned above. Within the promoter sequence will be the transcription initiation site, as well as the protein binding domains (consensus sequences) responsible for the RNA polymerase binding. Frequently, but not always, eukaryotic promoters contain "TATA" boxes and tables and "CAT". Prokaryotic promoters contain sec? Shine-Dalgarno in addition to the consensus sequences -10 ¡and -35. A "control expression sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A sequence that codifies this "under the control" of transcriptory and translational control sequences in a cell when 1 RNA polymerase transcribes the sequence encoding the mRNA, which is then translated into the protein encoded by the sequence that encodes. A "signal sequence" can be included near the sequence it encodes. This sequence encodes a signal peptide, N-terminal for the polypeptide, which I The term "primer" as used herein refers to an oligonucleotide, whether it occurs naturally m ** m *? m ^ ííMk The primers here are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers can be sufficiently complementary to hybridize with their respective strands. Therefore, the sequence of the primer does not need to reflect the sequence ^ Lyg & exact template. For example, a non-complementary nucleotide fragment may be linked in the final 5 'direction of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, the non-complementary bases or longer sequences may be interspersed in the primer, provided that the primer sequence has sufficient complementarity with the sequence or is hydrolyzed therein and thus forms the template for the synthesis of the product. of extension. i i I A cell has "transformed" DNA mediantje! heterologous or exogenous when such DNA has been introduced into the interior of the cell. The transforming DNA may or may not be integrated (covalently linked) into the cell genome. In prokaryotes, yeasts, and mammalian cells for example, the transforming DNA can be maintained in an episomal element such as a plasmid. With , > M- 'ú, «.- r-« Mfflfc »a» cells or clones that comprise a population of cells and daughters that contain the transforming DNA. A "clone" is a population of cells derived from a single cell or I progenitor by mitosis. A "cell line" is a clone i of a primary cell that is capable of establishing I I growth in vi tro by various generations. i i i Two DNA sequences are "substantially I I homologous" when at least approximately j 75% i (preferably at least about 80% and more preferably at least about 90% or 95%) of the nucleotides are paired at the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard count programs available in sequence data banks, or by low Southern hydridization experiments eg strict conditions defined by the particular system. Defining the appropriate hybridization conditions within those skilled in the art. See, for example, Maniatis et al., Supra; DNA Cloning, Volumes I & II, supra; Nucleic Acid Hybridization, supra.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the largest molecule in nature. Thus, when the region proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the commonly available procedures. The proto-isotope can be selected from H, C, 32P, 35S ,,? 36C, l, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131 I, and 186Re. Label enzymes are equally useful, and can be detected by any of the colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques currently used. The enzyme is conjugated to the selected particle by reaction with cross-molecules such as carbodiimides, diisocyanates, glutaraldehydes and the like. Various enzymes that can be used in these procedures are known and can be used. Preferred are: peroxidase, β-glucuronidase, ß-D-glucosidase, ß-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. The U.S. Patent Nos. 3,654,090, 3,850,752 and 4,016,043 are referred to by way of example for their description of alternative labeling methods and materials. ii As used here, the term "host" includes not only prokaryotes but also eukaryotes such as * i i,? mÉ .- *. »--_ ¿jfaMfc. Not even me? b? nt ^ a ^ yeasts, plant and animal cells. A molecule of i I DNA or gene encoding an immunoreactive protein of 28-kDa I I of Ehrlichia canis of the present invention can be used to transform a host using any of the techniques commonly known to those skilled in the art. Especially preferred is the use of a vector containing i sequences encoding a gene encoding I I a 28-kDa immunoreactive protein of Ehrlichia cafois of the present invention for transformation and prokaryotic purposes. ¡Prokaryotic guests can include E. coli, S. tymphimuri um, Serra tia marcescens and Ba cil l us subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells. In general, expression vectors containing promoter sequences that facilitate efficient transcription of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter (s), terminator (s), as well as specific genes that are capable of providing phenotypic selection in transformed i cells. The transformed hosts may be 3 or SEQ ID No. 5. The protein encoded by the DNA of this invention may share at least 80% of the sequence identity (preferably 85%, and more preferably 90%, and more preferably 95%) with the amino acids listed in SEQ ID No. 2 or SEQ ID No. 4 or SEQ ID No. 6. More preferably, the DNA includes the coding sequence of the nucleotides of SEQ ID. 1, from the same. Such a probe is useful for detecting the expression of the 28-kDa immunoreactive proteins of Ehrlichia canis in a human cell by a method that includes the steps of (a) contacting the mRNA obtained from the cell with the labeled hybridization test.; and (b) I detect the hybridization of the test with the mRNA. The invention also includes a substantially pure DNA containing a sequence of at least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the region of the nucleotides listed in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No.

'High severity' refers to DNA hybridization and washing conditions characterized by high temperature and low salt concentration, eg, washing conditions of 65 ° C at a salt concentration of approximately 0.1 x SSC, or the functional equivalent thereof. For example, high severity conditions may include I hybridization at about 42 ° C in the presence of about 50% formamide; a first wash to contains 1% SDS; followed by a second wash of approximately 65 ° C with approximately 0.1 x SSC. By "substantially pure DNA" is meant DNA that is not part of a medium in which DNA occurs naturally, by virtue of the separation (partial or partial purification) of some or all of the molecules in the environment, or by virtue of alteration of sequences flanking the claimed DNA. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into a virus or plasmid that replicates autonomously, or into the genomic DNA of a procari.ote or eukaryote; or that it exists as a separate molecule (eg, a genomic cDNA or a cDNA fragment produced by the polymerase chain reaction (PCR) or restriction of the endonuclease digestion process) independent of other sequences. It also includes a DNA The DNA can have at least about 70% of the sequence identity for the coding sequence of the nucleotides listed in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5 preferably at least 75% (eg, at least 80%); and more preferably at least 90%. The identity between the two sequences is a function directed to the number of identical matings or positions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, for example, if a given position is occupied by an adenine in each of the two DNA molecules, then these are identical to the position. For example, if 7 positions in a sequence of 10 nucleotides in their length are identical to the corresponding positions in a second sequence of 10 nucleotides, then, the two sequences have 70% sequence identity. The length of the comparison sequences will generally be at least 50 nucleotides, and preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and more preferably 100 nucleotides. Identity is typically measured using a sequence analysis (eg, Software Package of the Genetics Computer Group, Uniyersity of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). The present invention comprises a vector comprising a DNA sequence coding for a gene! encoding a 28-kDa immunoreactive protein of Ehrli chia canis and said vector is capable of replicating in a host comprising, in an operable union: a) an origin of replication; b) a promoter; and c) a sequence of DNA coding for said protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. 5. A "vector" can be defined as an acid term replicable nucleic acid, eg, a plasmid or viral nucleic acid. The vectors can be used to amplify and / or express nucleic acids encoding a 28-kDa immunoreactive protein of Ehrlichia canis. An expression vector is a replicable term in which the amino acid sequence encoding a polypeptide is operably linked to the appropriate control sequences I capable of effecting the expression of the Jen polypeptide in the cell. The need for such control sequences will vary depending on the cell selected the selected transformation method. Generally, the control sequences include a transcriptional promoter and / or enhancers, suitable for the ribosomal binding site mRNA, the sequences that control the termination of transcription and translation. Methods that are well known to those skilled in the art and can be used to construct expression vectors that contain appropriate transcriptional and transcriptional control signals. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A labora tory Manual (2nd Ed.), Cold Spring Harbor Press, N. Y. A gene and its transcriptional control sequences are defined as "operably linked" if the transcriptional control sequences effectively control the transcription of the I gene. Vectors of the invention include, but are not limited to, vectors of plasmids and viral vectors. The preferred viral vectors of the invention are those! derivatives of retroviruses, adenoviruses, adeno-associated viruses, SV40 virus or herpes virus. By a "substantially pure protein" is meant a protein that has been separated from at least some of those components that naturally accompany these. Typically, the protein is substantially pure j ^^ jj when at least 60% by weight, is free of proteins and weight. A substantially pure 28-kDa immunoreactive protein of Ehrlichia canis can be obtained, e.g., by extraction of a natural source; by means of the A protein is substantially released from the naturally associated components when these are separated from at least some of those contaminants that accompany these in their natural state. Thus, a protein that is synthesized I chemically or produced in a cellular system different from I naturally associated Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, or any other organism in which they do not occur naturally. In addition to the full length or full length proteins, the invention also includes fragments (eg, antigenic fragments) of the 2! immunoreactive kDa of Ehrlichia canis (SEQ ID No. 2 or ISEC ID I No. 4 or SEQ ID No. 6). As used herein, "fragmented" as applied to the polypeptides will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (eg, 50 residues) in length, but less than the intact sequence. total. Fragments of the immunoreactive 28-kDa protein of Ehrlichia canis can be generated by methods known to those skilled in the art, for example, by the enzymatic digestion process of the recombinant Ehrlichia canis immunoreactive 28-kDa protein or that occurs naturally, by recombinant DNA techniques using an expression vector encoding a defined fragment of an immunoreactive 28-kDa protein from Ehrl i chia canis, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of the immunoreactive 28-kDa protein of Ehrlichia canis (for example, binding to an antibody specific for the immunoreactive 28-kDa protein of Ehrlichia canis) can be evaluated by methods described herein. The immunoreactive 28-kDa protein of purified Ehrlichia canis or antigenic frgments of the 128-kDa immunoreactive protein of Ehrlichia canis can be used to generate new antibodies or to test existing antibodies (eg, as positive controls in a test of diagnosis) using standard protocols by those skilled in the art. Included in this invention is the polyclonal antiserum generated by the use of the immunoreactive 28-kDa Ehrli chia canis protein or a fragment of the immunoreactive 28-kDa protein of Ehrlichia canis as the immunogen in e.g., cqnejos. Standard protocols for the production of the monoclonal and polyclonal antibody known to those skilled in the art are employed. The monoclonal antibodies i generated by this method can be protected by the ability to identify recombinant Ehrlichia canis clones, and to distinguish them from known cDNA clones. "..i,. , t__J_¿t ____ > tl iriiii f ^ ^ t¡¿¿ß Additionally included in this invention are fragments of the immunoreactive 28-kDa protein of Ehrlichia canis that are encoded at least in part by portions of SEQ ID No. 1 or SEQ ID No. 3 or SEQ ID No. I 5, eg, splicing products from! Alternative mRNA 1 or events that process alternative protein, or in which a section of the sequence has been deleted.

The fragment, or the immunoreactive 28-kDa protein of Ehrli chia canis intact can be covalently linked to another polypeptide, for example., Which act as a label, a ligand or a means of increasing antigenicity. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a human. The preparation of an aqueous composition containing a protein as an active ingredient is well understood in the art. Typically, such compositions i are prepared as injectables, either as liquid solutions or suspensions; Suitable solid forms for the solution in, or suspension in, may also be liquid preparations prior to injection. The preparation can also be emulsified.

A protein can be formulated in a composition of a salt or neutral form. Pharmaceutically acceptable salts include the addition of acid salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic , and similar. Salts formed with free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. After the formulation, the solutions will be administered in a manner compatible with the formulation of the dose and in therapeutically effective amounts. The formulations are easily administered in a variety of dosage forms such as injectable solutions. For administration other than parenterally in an aqueous solution, for example, the solution should be adequately buffered if necessary and the diluent liquid first supplied isotonic with sufficient saline or glucose. These rich solutions ^ J * »particular are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administrations. In connection with this, the sterile aqueous medium that can be employed will be known to those skilled in the art in the clarity of the description.

I presented. For example, a dose could be dissolved in 1 ml of an isotonic NaCl solution and either 1000 ml added! of fluid hypodermoclysis or injected into the planned site of preparation, (see for example, "Remington's I Pharmaceutical Sciences" Fifteenth edition, pages 1035-1038 and 1570-1580). Any variation in the dose will necessarily be presented depending on the condition of the subject in question. The person responsible for the administration will depend on any event to determine the appropriate dose for a particular subject. As is well known in the art, a given polypeptide is often immunogenic (by invention) with preferred are albumin of sue a variety of lymphokines and adjuvants such as IL2, 114, IL8 and others. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbo-diimide and bis-biazotized benzidine. It is also understood that the peptide can be conjugated to a protein by genetic engineering techniques that are well known in the art. ! As is also well known in the art, the immunogenicity for a particular immunogen can be improved by the use of non-specific stimulators of the immune response known as adjuvants. Examples of preferred adjuvants include complete BCG, Detox, (RIBI, Immunochem Research Inc.) ISCOMS and aluminum hydroxide adjuvant (Superphos, Biosector). As used herein, the term "complement" is used to define the nucleic acid strand that will hybridize to the first nucleic acid sequence to form a double-stranded molecule under stringent conditions. The stringent conditions are those that allow hybridization between the two nucleic acid sequences with a high degree of homology, but not including The temperature and ionic strength of a desired severity are understood to be applicable to the lengths of the particular tests, for the base content and sequence sequence and for the presence of formamide in the hybridization mixture. As used herein, the term "genetically engineered" or "recombinant" cells is intended to refer to a cell in which the recombinant gene, i has been introduced such as a gene encoding an antigen of Ehrli chia chaffeensis. Therefore, cells generated by genetic engineering are distinguished from naturally occurring cells which do not contain, a gene introduced recombinantly. Thus, the cells generated by genetic engineering are cells that have a gene or genes introduced either in the form of a gene of i cDNA, a copy of a genomic gene, or will include genes positioned adjacent to a naturally associated non-associated promoter. with the gene introduced particularly. In addition, the recombinant gene can be integrated into the genqma of the MMéak-M-bÜH-My host, or it can be contained in a vector, or in a bacterial genome transfected in the host cell. The following examples are given for the purpose of illustrating various embodiments of the invention and are not intended to limit the present invention in any style.

EXAMPLE 1 Ehrlichiae and i Purification! ' ! The Ehrli chia canis (strain Florida and isolated Demon, i DJ, Jake, and Fuzzy) are provided by Dr. Edward Breitschwerdt, (College of Veterinary Medicine, 'North Carolina State University, Raleigh, NC). E. canis * (strain Louisiana) was provided by Dr. Richard E. Córstvet (School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA) and £. canis (Oklahoma strain) was provided by Dr. Jacqueline Dawson (Centers for Disease Control and Prevention, Atlanta, GA). Propagation of I ehrlichiae was performed in DH82 cells with DMEM supplemented with 10% bovine calf serum and 2 mM 1-glutamine at 37 ° C. The intracellular growth in DH28 cells was monitored by the presence of a morula of £. canis using general methods of cytological staining.

The cells were harvested when 100% of the cells were ~ ^ * ^ were infected with ehrlichiae and pellets were then formed in a centrifuge at 17,000 x g for 20 minutes. The cell pellets were destabilized with a sonicator Braun-Sonic 2000 twice at 40W for 30 seconds on ice. The Ehrlichiae was purified as previously described (Weiss et al., 1975). The lysate was loaded on discontinuous gradients of 42% -36% -30% renografin, and centrifuged at 80,000 x g for 1 hour. The heavy and light bands containing ehrlichiae were harvested and washed with sucrose-phosphate-glutamate buffer (SPG, 218 mM sucrose, 3.8 mM KH2P04, 7.2 mM K2HP04, 4.9 mM glutamate, pH 7.0) and pellets were formed by centrifugation.

I Nucleic Acid Preparation L Ehrlichia canis DNA was prepared by resuspension of purified ehrlichiae with renografin in 600 μl of 10 mM Tris-HCl buffer (pH 7.5) with 1% sodium dodecyl sulfate (SDS, weight / volume) and 100 ng / ml protein K as previously described (McBride et al., 1996). This mixture was incubated for 1 hour at 56 ° C, and the nucleic acids were extracted twice with a mixture of phenol / chloroform / isoamyl alcohol (24: 24: 1). The DNA is It was pelleted by absolute ethanol precipitation, washed once with 70% ethanol, dried and resuspended in lOmM Tris (pH 7.5). The plasmid DNA was purified using a High Isolation Plasmid kit.

Purity (Boehringer Mannheim, Indianapolis, IN), and the! PCR products were purified using a QIAquick PCR Purification Kit (Qiagen, Santa Clarita, CA).

EXAMPLE 3 PCR amplification of the 28-kba E protein genes. ¡I canis i The regions of 1 gene of E. canis ECa28-l Jotun-Hein of E. p28 chaffeensis and the Cowdria ruminant tí um map-1 genes. The forward primer 793 ((5- I GCAGGAGCTGTTGGTTACTC-3 ') (SEQ ID No. 16) and reverse primer 1330 (5'-CCTTCCTCCAAGTTCTATGCC-3 ') (SEQ ID No. 17) corresponding to nucleotides 313-332 and 823-843 < of C.! Ruminate ti um MAP-1 and 307-326 and 834-814 of E. chaffeensis P28. 1 E. canis (a North Carolina isolator, Jake) the DNA was amplified with primers 793 and 1330 with a thermal cyclization profile of 95 ° C for 2 minutes, and subjected to 30 cycles of 95 ° C for 30 seconds, 62 C for 1 minute, 72CC for 2 minutes followed by an extension of 72 ° C for 10 minutes and 4 ° C. The PCR products were analyzed in 1% agarose gels. The amplified PCR product was sequenced directly with primers 793 and 1330. i The primers specific for the ECa28SA2 gene designated 46f (5'-ATATACTTCCTACCTAATGTCTCA-3 ', SEQ ID No. 18) and primer 1330 (SEQ ID NO. 17) were used to amplify the target region. The amplified product was gel purified and cloned into a TA I cloning vector (Invitrogen, Santa Clarita, CA). The clone was bidirectionally sequenced i with primers: inverse M13 of the vector, 46f, Eca28SA2 (5'- AGTGCAGAGTCTTCGGTTTC-3 ', SEQ ID No. 19) ECa5. 3 (5'-GTTACTTGCGGAGGACAT-3 ', SEQ ID No. 20). The DNA was amplified with a subject thermal cyclization profile 95 ° C for 2 minutes, and 30 cycles of 95 ° C for 30 seconds, 48 ° C for 1 minute, 72 ° C for 1 minute followed by an extension of 72 ° C for 10 minutes and 4 ° C.

EXAMPLE 4 Unknown Sequencing of the 3 'and 5"Regions of the Eca28-1 Gene The total length of the sequence of Eca28-1 was determined using a Universal Genome Walker device (CLONTECH, Palo Alto, CA) according to the protocol supplied by the manufacturer. The genomic DNA of E. canis (Jake isolate) was digested completely with five restriction enzymes (Dral, EcoRV, PvuII, Seal, Stul) that produces DNA finished in edge. A power adapter (API) in the equipment was ligated to the end of the E DNA. canis. Genomic libraries were used as templates to find the known DNA sequence of the ECa28-l gene by PCR using a primer complementary to a known portion of the ECa28-l sequence and a specific primer for the API adapter. Primers specific for ECa28-l used for the walking genome were designed from the known DNA sequence derived from PCR amplification of ECa28-l with primers 793 (SEQ ID No. 16) and 1330 (SEQ ID NO. 17). The primers 394 (5'-GCATTTCCACAGGATCATAGGTAA-3 '; nucleotides 687-710, SEQ ID No. 21) and 394C (5'-TTACCTATGATCCTGT GGAAATGC-3; NUCLEOTIDES 710-687, SEQ ID No API provided known from the corresponding gene to the 5 'region of the amplified ECa28-l gene with primers 394C and API (2000-bp) were sequenced unidirectionally with the 793C primer (5'GAGTAACCAACAGCTCCTGC-3 ', SEQ ID No. 23). A PCR i i product corresponding to the 3 'region of the ECa28-l gene amplified! with primers 394 and API (580-bp) were sequenced i I bidirectionally with the same primers. The non-coding regions ii in the adjacent 3 'and 5' regions and the open reading frame were sequenced, and the primers EC280M-F (5'- TCTACTTTGCACTTCC ACTATTGT-3 ', SEQ ID No. I 24) AND EC280M-R (5'-ATTCTTTTGCCACTATTT TTCTTT-3 ', SEQ ID I I No. 25) complementary to the regions were designed to amplify the entire ECa28-l gene.

EXAMPLE 5, ~ - ^ - ~~~ - I I Sequencing of E. canis isolates The DNA was sequenced with an ABI Prism 377 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). The whole genes of seven isolated ECa28-l (four from North Carolina, and each from Oklahoma, Florida and Louisiana) were amplified by PCR with primers EC280M-F (SEQ ID No. 24) and EC280M-R (SEQ. ID No. 25) with a thermal cyclization profile of 95 ° C for 5 minutes, and 30 cycles of 95 ° C for 30 seconds, 62 ° C for 1 minute, and 72 ° C for 2 minutes and an extension of 72 ° e for 10 minutes. The resulting PCR products are bidirectionally with the same primers.

EXAMPLE 6 Cloning and Expression of E, canis ECa28-l i The E gene. canis ECa28-l integrated was amplified with PCR with primers EC280M-F and EC280M-R and cloned into a cloning vector pCR2.1-TOPO TA to obtain the desired set II of the restriction enzyme cleavage sites (Invitrogen, Carlsbad, CA). The insert was excised from pCR2.1-TOPO with BstX 1 and ligated into the eukaryotic DNAse 3.1 expression vector (Invitrogen, Carlsbad, CA) designed for cDNA 3.1 / EC28 for subsequent studies. Plasmid DNApc 3.1 / EC28 was amplified, and cleaved with a double digestion of Kpnl-Xbal and a prokaryotic expression vector pThioHis (Invitrogen,! Carlsbad, CA) was ligated directionally into I. The clone (designed pThioHis / EC28) produced a recombinant thioredoxin fusion protein in Escherichia coli BL21. the recombinant fusion protein was crudely purified in the insoluble phase by centrifugation. The control thioredoxin fusion protein was purified from cell phones under native conditions using turning columns of NTA with nickel (Qiagen, Santa Cljarita, I CA). ! i I I I I EXAMPLE 7 Western Immunoassay Assay The recombinant fusion protein E. caní s \ ECa28- 1 was subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) in 4-15% gradient gels Tris-HCl (Bi-o-Rad, i Hercules, CA) and was transferred to pure I nitrocellulose i (Schleicher &Schuell, Keene, NH) using a cell from semi-dry transfer (Bio-Rad, Hercules, CA). The membrane was incubated with the antiserum of the convalescent phase of a Poerro infected with E. canine diluted 1: 5000 for 1 hour, washed, and then incubated with a secondary antibody from purified affinity alkaline conjugated phosphatase (H & L) anti-canine IgC 1 at 1: 1000 for 1 hour (Kirkegaard &Perry Laboratories, Gaithersburg, MD). The bound antibody is visualized with substrate 5-bromo-4-chloro-3-yl phosphate / nitro blue tetrazolium (BCIP / NBT) (Kirkegaard &Perry i i Laboratories, Gaithersburg, MD). 1 I I EXAMPLE 8 i Southern blot analysis a - Éi -_--- i-IÍ_áu < ... * Í- **. *. *. -., * > ,. *, * > . . «. IX.A To determine the multiple homologous genes for the Eca28-1 gene that were presented in the E. canis genome, a genomic Southern blot analysis was performed using a standard procedure (Sambrook et al., 1989). The genomic DNA of E. canis was completely digested with each of the restriction enzymes Bail, EcoRV, Hael l, Kpnl and? Spel, I that does not excise with the ECa28-l gene, and Asel that digests the i I nucleotides ECa28-l 34, 43 and 656. The test was analyzed ii by PCR amplification with primers EC280M-F and I 10 EC28M-R and deoxynucleotide triphosphates labeled with (DIG) II digoxigen (dNTPs) (Boehringer Mannheim, Indianapolis,! IN) and i digested with Asel. The digested test (566-bp) was separated by agarose gel electrophoresis, purifixed gel and then used by hybridization. The DNA of E. canis completely digested was electrophoresed and transferred to a nylon membrane (Boehringer Mannheim, Indianapolis, IN) and hybridized at 40 ° C for 16 hours with the DIG-labeled test of the ECa28-l gene in Hyb Easy i DIG buffer according to the manufacturing protocols (Boehringer Mannheim, Indianapolis, IN). The bound test was detected with an anti-DI3 anti-alkaline phosphatase antibody and a luminescent substrate (Boehringer Mannheim, Indianapolis, - * "... .... **. Í? U¿ ?. ". *.« **. i - ^ .. ^^.,. .,,. ^^ g ^^ ¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡) and was exposed to a scientific image film iBioMax (Eastman Kodak, Rochester, NY).

EXAMPLE 9 Sequence Analysis and Comparison 'I The DNA sequences of C. ruminan ti um map-1 and E. i chaffeensis p28 were obtained from the National Center l of I I Information on Biotechnology (NCBI) (World Wide Web site at URL: http: // ww. Ncbi. Nlm. Nih. Gov / Entrez). The nucleotide and I amino acid sequences, the phylogenetic analysis and the protein i were performed with a LASERGENE computer program (DNASTAR, Inc., Madison, Wl). The analysis of the translational post-I process was carried out using the McGeoch and von Heijne method by means of signal sequence recognition using the SPORT program (Mcgeoch, 1985, von Heijne, 1986) (World Wide Web site at URL: PRÍVATE HREF = "http // www imcb osaka-u.ac.jp/nakai/form.htm", MACROBUTTON HtmlResAnchor http: // www. Í cb. Osaka-u.ac.p / nakai / form. Htm). I The access numbers for the nucleic acid and I amino acid sequences of the ECa28-l genes of E. 'Canis describe in their study: Jake, AF082744; siana AF082745; Oklahoma, AF082746; Demon, AF082747; DJ, 82748; Fuzzy, AF082749; Florida, AFO82750.

* ¡? A? *? * A * m * - ^^ mm ^^ * * a ^ * lláß ^ áamlm The sequence analysis of ECa28-l of | seven different strains of E. canis were made with primers designed to extend the whole gene. The analysis revealed that the sequence of this gene was conserved between the insulators of North Carolina (four), Louisiana, Florida and Oklahoma.

EXAMPLE 10 Amplification by PCR, Cloning, Sequencing and Expression of Eca28-1 Alignment of the nucleic acid sequences of E. chaffeensis p28 and Cowdpa ruminantium map-1 using the Jotun-Hein algorithm produced a consensus sequence with I regions of high homology (>90%). These hoological regions (nucleotides 313-326 and 814-834 of E. chaffeensis p28) were the abject sites as hybridization sites by heating and cooling of primers for PCR amplification. PCR amplification of the genes E. I canis ECa28-l and E. chaffeensis p28 was completed with I I primers 793 and 1330, resulting in a PCR product of 518-bp. The nucleic acid sequence of the PCR product, from E. i canis was obtained by sequencing the pipeline directly with primers 793 and 1330. The analysis of the sequence revealed an open reading structure that - * ^^ encodes a protein of 170 amino acids, and the alignment of! the sequence of 518-bp obtained from the PCR amplification of E. canis with the DNA sequence of the gene E. chaffeensis p28 revealed a greater similarity than 70%, indicating that the genes are homologous. The PCR adapter with the primers 394 and 793C were made to determine the sequence of the i I segments 5 'and 3' of the integral gene. Primer 394 produced four PCR products (3-kb, 2-kb, 1-kb, and 0.8-kb),! and the 0.8-bp product was sequenced bidirectionally using primers 394 and API. The deduced sequence overlapped j with the 3 'end of the product 518-bp, extending the open reading structure of 12-bp to a termination codon. A 1 sequence that does not encode additional 625-bp in the final 3'j of the ECa28-l gene was also sequenced. Primer 394C was used to extend the final 5 'of the ECa28-l gene with the API supplied primer. Amplification with these primers resulted in three PCR products (3.3, 3-kb, and 2-kjb). The 2-kb fragment was sequenced unidirectionally with primer 793C. The sequence provided the putative start codon of the ECa28-l gene and was completed by open reading 834-bp encoding a 278 amino acid. An additional i 144-bp readable sequence was generated in the 5 'non-coding region of the ECa28-l gene.

The primers EC280M-F and EC2 (= MR were designed from the complementary non-coding regions adjacent to the i ECa28-l gene.) The PCR product amplified with these first 1 primers was sequenced directly with the same primers. complete DNA sequence (SEQ ID No. 1) for the 1 gene ECa28-l of E. canis is shown in figure 1. The PCR fragment of ECa28-l was amplified with these primers contained in the open reading frame whole and 17 j amino acids of the 5 'non-coding primer region.The I gene was directionally subcloned into the expression vector pThioHis, and E. coli (BL21) was transformed into its construct.The thioredoxin fusion protein ECa28- The expressed protein was insoluble, the protein expressed has 114 amino acids associated with the thioredoxin, 5 amino acids for the enterokinase recognition site, and 32 amino acids from the multiple cloning site and the primer region. or coding 5 'in the term N. The convalescent phase of the antiserum of a dog infected by E. canis recognized the recombinant fusion protein expressed, but does not react with thioredoxin control. (Figure 2). i EXAMPLE 11 I Sequence Homology i The nucleic acid sequence of ECa28-l (8¡34-bp) i and the family of E genes. Chaffeensis omp-l that injclude! Signal sequences (ECa28-l, omp-lA, B, C, D, E, and F) were aligned using the Clustl method to examine the homology between these genes (no alignment shown). Nucleic acid homology was similarly conserved (68.9%) between i ECa28-l, and E. chaffeensis p28 and omp-l. Other putative outer membrane protein genes in the E family. chaffeensis omp-l, omp-lD (68.2%), omp-lE (66.7%), omp-lC (64.1%), Cowdria ruminate ti um map-1 (61.8%), 1 g of protein of 28 -kDa E. canis (60%) and 2 protein genes of 28-kDa (partial) (59.5%) are also homologous to ECa28-l. E. chaffeensi s omp-lB has at least nucleic acid homology (45.1%) with ECa28-l. The alignment of the predicted amino acid sequences of ECa28-l (SEQ ID No. 2) and E. P28 chaffeensis reveal amino acid substitutions resulting from I four variable regions (VR). Substitutions or deletions in the amino acid sequence and the locations of i I the variable regions of ECa28-l and the family E. chaffeensis OMP-1 were identified (Figure 3). The amino acids compared to the signal peptide revealed that I Eca28-1 shares the highest homology with OMP-1F (68%) i of the family E. chaffeensis OMP-1 followed by E. chaffeens \ is P28 (65.5%), OMP-1E (65.1%), OMP-1D (62.9%), O%), Cowdria ruminate tum MAP-1 (59.4%), 1 protein of 28-kDa E. i canis (55.6%) and 2 proteins of 28-kDa (partial) (53.6%), and OMP-1B (43.2%). The phylogenetic relationship based on the! Amino acid sequences show that ECa28-l 'and C. Ruminantium MAP-1, proteins E. chaffeensis OMP-1, and 1 protein 28-kDa of E. canis and 2 (partial) are related (Figure 4).

EXAMPLE 12 Probability of Predicted Surface and Immunoreactivity The analysis of Eca28-1 of E. canis using hydropathic profiles and hydrophilicity profiles in the exposed surface junctions predicted in Eca28-1 (Figure 6). | The eight major exposed surface regions that I consist of 3 to 9 amino acids were identified in Eca28-1 and are similar for the profile of the regions exposed to the surface in E. chaffeensi s P28 (Figure 6). Five, the 1 large regions exposed to the surface in Eca28-1 were located in the N-terminal region of the protein. The aM ^ a | ^^ kiß | | j | ^ H ¡^ ari | ji |||| HAM || aa m ^ ^^? Jlg i hydrophilic regions exposed to the surface were found in all four variable regions of Eca28-1. 10 portions of T cells were predicted in E? ca28-1 i using the Rothbard-Taylor algorithm (Rothbard and Tjaylor, I 1988 ), and high antigenicity of Dca28-1 was predicted by the Jameson-Wolf antigenicity algorithm (Fig. 6) (Jameson and Wolf, 1988). Similarity was observed in the! I antigenicity and the portions of T cells between Eca28-1 and I E. chaffeensis P28. EXAMPLE 13 Detection of the homologous genomic copies of the Eca28-1 gene. The analysis of Southern genomic spotting of E. canis DNA i digested completely independently with the restriction enzymes Bañil. EcoRV, Haell, Kpnl, Spel, 1 restriction endonuclease in the Eca28-1 gene, and Asjel, which has internal restriction endonuclease sites at nucleotides 34, 43 and 656, revealed the presence of at least I three copies of Eca28-1 gene homologs (Figure 5). Although Eca28-1 has internal restriction sites, the 1 probe labeled with DIG used in the I hybridization experiment targets a region of the gene within a single DNA fragment generated by the digestion of • * "* • -" - -T- * * * - ** - »». * &? < i? Asel of the gene. Digestion with the Asel produced 3 'bands (about 566-bp, 850-bp and 3-kb) Hybridized with the DNA probe Eca28-1 indicated the presence of multiple gene homologs to Eca28-1 in the genome. The ! digestion with EcoRV and Spel produced two bands that hybridized with the Eca28-1 gene probe. EXAMPLE 14 Identification of the Place of the 28-kDa Protein Gene. The specific primers designated i EcaSA3-2 (5 '-CTAGGATTAGGTTATAGTATAAGTT-3', SEQ ID No. 26) corresponding to the Eca28SA3 regions and the primer 793C were used. (SEQ ID No. 23) that is tempered to a region with Dca28-1 To extend the intergenic region between the SA3 gene and ^ Eca28-1. The 800b-p product was sequenced with the same primers. The DNA was amplified with a thermal cyclization profile of 95 ° C for 2 minutes, and 30 cycles of 95 ° C for 30 seconds, 50 ° C for 1 minute, 72 ° C lasting 1 ° 1 minute followed by an extension 72 ° C was maintained for 10 minutes and 4 ° C.

EXAMPLE 15 i The PCR Amplification of the 2i kDa Protein Genes and the Identification of the Place of Multiple Genes < Mt »?? *? «Mtíit.a .... - - ii > In order to specifically amplify II the known genes in the 3 'direction of ECa28SA2, the 46f primer for ECa28SA2, and primer 1330 was used which is directed to a region conserved at the end 3 'of the ECa28-l gene for amplification A 2-kb PCR product was amplified with these primers containing 2 open reading frames The first open reading frame comprises the known region of the gene, ECa28SA2, and a 3 'portion of the gene not previously sequenced In the 3' direction of an additional ECa28SA2, a 28kDa protein gene was found that was homologous but not identical and was designed Eca28SA3. The two known sites were joined by amplification with the SA-2 primer specific for the 3 'end of the Eca28SA3 gene were used in conjunction with a reverse primer 793C, which is hybridized by the application of heat and cooling at the end of ECa28-l. An I-PCR product of 800-bp was amplified at the end! 3 'of Eca28SA3, the intergenic region between Eca28SA3 and i ECa28-l (28NC3) and the 5' end of ECa28-l, which was bound to the previously separated site (Figure 8). The 849-bp open reading frame of ECa28SA2 encodes a prp 1 teine of 283 amino acids, and the Eca28SA3 has an open reading structure of 840-bp that encodes a protein of 280 í «É-lÍÍMÍ-rt-MÍM amino acids. The non-coding intergenic region between Eca28SA3 and ECa28-l was 345. bp in length (Figures 7 and EXAMPLE 16! Homology of Amino Acid and Nucleic! The amino acid and nucleic sequences of all five genes of the 28-kDa protein were aligned using the Clustal method to examine the homology between these genes. Nucleic acid homology was found in the range of 58 to 75% and similar amino acid homology was observed in a range of 67 to 72% between the members of the 28-kDa protein gene of E. cani s (Figure 9). I EXAMPLE 17 Transcriptional Promoting Regions The intergenic regions between the ¡de genes of the 28-kDa protein were analyzed for the promoter sequences by comparing the promoter I 1 promoter Escherichia coli regions and a promoter] of E. chaffeensis (Yu et al., 1997 McClure, 1985). The putative 1 1 promoter sequences including RBS, regions -10 and -35 'in 4 were identified. intergenic sequences corresponding to the i genes ECa28SA2, Eca28SA3, ECa28-l, and ECa28-2 (Figure 10). The non-coding region in the 5 'direction of ECa28SAl < it is not known and it was not analyzed.

EXAMPLE 18 N-terminal Signal Sequence The analysis of the amino acid sequence revealed that the entire E. canis Eca28-1 has a molecular mass! deduced from 30.5-kDa and the entire Eca28SA3 has a mass molecular weight of 30.7-kDa. You love proteins have an N-terminal signal peptide of 23 amino acids (MNCKKILITTALMSLMYYAPSIS, i SEQ ID No. 27), which is similar to that predicted by E. chaffeensis P28 (MNYKKILITSALISLISSLPGV SFS, SEQ ID No. 28), and the OMP-1 protein family (Yu et al., 1998; Ohashi et al. ., 1998b). A preferred cleavage site will stop signal peptidases (SIS, Ser-X-Ser) (Oliver, 1985) found in amino acids 21, 22, and 23 of ECa28-l. An additional putative cleavage site I was also presented at the position of amino acid '25 (MNCKKILITTALISLMYSIPSISSFS, SEQ ID No. 29) identical to the predicted cleavage site of E. chaffeensis P28 (SjFS), and 1 result in a mature Eca28-1 with a molecular mass n.t i? i.i.irr? • i., - i, r i ^ e-aMMÉjÉt-l ^ iiÉÉMi ^ ^ predicted of 27.7-kDa. The cleavage site I i was predicted from the partial sequence previously reported from Eca28SA2 at amino acid 30. However, the analysis of the predicted signal sequence had an unscreened signal sequence.

Summary Proteins of similar molecular mass have been identified and cloned from multiple riches agents including E. canis, E. chaffeensis, > and C. I ruminantium (Reddy et al., 1998; Jongejan et al., < 1993; Ohashi et al., 1998). A single site has been previously described in Ehrli chia chaffeensis with 6 p28 homologous genes, and 2 places in E. canis containing some genes i of the 28-kDa protein. The present invention demonstrated that the cloning, expression and characterization of the genes encoding a mature 28-kDa protein from E. canis that are homologous to the omp-l multiple gene family of E. chaffeensis and the I i C gene ruminate ti um. Two new genes were identified from the 28-kDa protein, ECa28-l and Eca28SA3. Another I-protein gene of 28-kDa E. canis, ECa28SA2, partially sequenced previously (Reddy et al., 1998), was completely sequenced in the present invention. The identification and characterization of a single place in E is also described. canis that contains all the five genes of the protdine of I 28-kDa E. canis. The 28-kDa E. canis protein is homologous to the I I family E. OMP-1 chaffeensis and the MAP-1 protein of C. i rumanin tium. The majority of homologous proteins of 28-kDa! E. canis (Eca28SA3, Eca28-1 and Eca28-2) meet! in the sequential range of the place. The homology of these proteins I are in the range of 67.5% to 72.3%. the divergence between these 28-kDa proteins was from 27.3% to 38.6%. the 28-kDa proteins E. canis, Eca28SAl and Eca28SA2 were at least homologous with a range of homology of 50.9% to 59.4% and I! divergence from 53.3 to 69.9%. The differences between the genes lie mostly in the four regions and it is suggested that these regions be exposed on the surface and the subject select the pressure by the immune system. The conservation of ECa28-l has been reported among the seven isolated E. canis (McBride et al., 1999). Suggesting that the E. I canis may be clonal in North America. Conversely, 1 significant diversity of p28 has been reported among isolated E. I chaffeensis (Yu et al., 1998). All proteins of 28-kDa E. canis are I I presented by being post-translational processed from _ ^ ^^^ _ ^^ _ ^^^^^^^^^ * ¿^^^^^^^^^^^^^ A ^^^^^ < A- »MSy.J? .tf I! II a 30-kD protein to a mature 28-kD protein, ii Recently, a signal sequence was identified in E. I chaffeensis P28 (Yu et al., 1998) and the N-terminal amino acid sequence has verified that the protein is processed post-translationally resulting in the cleavage of II the signal sequence to produce a mature protein (Ohashi et al., 1998). The leader sequences of 0MP-1F 'and OMP-1E have also been proposed as leader signal peptides.

(Ohashi et al., 1998). The signal sequences identified I in E. chaffeensis OMP-1F, 0MP-1E and P28 are homologous! to the leader sequences of the 28-kDa protein E. canis. The promoter sequences for the p28 genes have not been determined experimentally, but the putative promoter regions were identified by comparing the consensus sequences of the RBS, the promoter regions 1-10 and -35 of E. coli and other ehrlichiae (Yu et al.,: 1997); McClure, 1985). Such promoter sequences would allow each gene to be potentially transcribed and translated,! I suggesting that these genes can be differentially expressed in the host. The persistence of infection in dogs can be related to the differential expression of p28 genes resulting in changes ; j .¿., - Att -! ^ y ^ ^^ antigenic in vivo, thus allowing the body to evade the immune response. The 28-kDa E. canis proteins may be important in immunoprotective antigens. Several reports have shown that the 30-kDa antigen of E. Canis exhibit strong immunoreactivity (Rikihisa et al., I 1992). The antiserum antibodies of the convalescent phase of humans and dogs have reacted consistently with proteins in this rangq size of E. chaffeensi s and E. canis, suggesting that these may be important immunoprotective antigens (Rikihisa et al., 1994, Chen et al., 1994, Chen et al., 1997). In addition, antibodies 30, 24 and 21-kDa proteins recently developed in the immune response for E. canis (Rikihisa et al., 1994, Rikihisa et al., 1992), suggesting that these proteins can be especially important in immune responses in the acute state of the disease. Recently, a family of homologous genes that encode outer membrane proteins with molecular masses of 28-kDa in E. chaffeensis, and mice immunized with E, have been identified. Recombinant P28 chaffeensis are presented by having developed immunity against the homologous change i I (Ohashi et al., 1998). It has been shown that P281 of E. chaffeensis is present in the outer membrane, and immunoelectron microscopy has located P28 'on the surface of the organism, and thus suggest that it can' serve as an adhesive (Ohashi et al., 1998). It is likely that the 28-kDa proteins of E. canis identified in this study have the same place and possibly serve as a similar function. , Comparison of different strains of ECa28-l from E. canis reveal that the gene is fully conserved. Studies involving E. chaffeensis have shown molecular and immunological evidence of diversity in the ECa28-l. Patients infected with E. chaffeensis [have variable immunoreactivity for 29/28-kDa proteins, 1 suggesting that this is antigenic diversity (Chen et al., 1997). Recently molecular evidence has been generated to support antigenic diversity in the p28 ide E gene. chaffeensis (Yu et al., 1998). A comparison of five insulating E. chaffeensis revealed that two isolates (Sapulpa and St. Vincent) were 100% identical, but three others (Arkansas, Jax, 91HE17) are divergent by no less than 13.4% at the amino acid level. The conservation of ECa28-l suggests that E. canis strains found in The United States can be genetically identical, and so the ^^ Mútí iá protein of 28-kDa of E. canis is an attractive candidIato of the vaccine for canine ehrlichiosis in the United States. i In addition, the analysis of E. canis isolates outside the United States can provide information regarding the origin and evolution of E. canis. The conservation of the 28-kDa proteins make an important potential candidate for a reliable serodiagnosis of canine ehrlichiosis. I The role of homologous genes is not conjoined at this point; however, the persistence of E infections. canis in dogs could conceivably be related to an antigenic variation due to the variable expression of the homologous genes of the protein 28-kDa, thus allowing E. canis to evade immune supervision. The variation of the msp-3 > in A.

Margina is partially responsible for the variation in the MSP-3 protein, resulting in persistent infections I (Alleman et al., 1997). Studies to examine the I expression of the 28-kDa protein gene by E. canis in chronically infected dogs and acutely I would provide insight into the role of the family of the 28-kDa protein gene in the persistence of the infection. ' ..... i-? ^ te *.

The following references are cited here. Alleman A.R., et al., (1997) Infect Immun 65: 156-163 Anderson B.E., et al., (1991) J Clin Mi crobiol 29: 2838¡-2842 Anderson B.E., et al., (1992) Int J. Syst Bacteriol 42 :, 299- i 302. 'Brouqui P., et al., (1997) Clin Diag Lab Immunol 4: 731-735. I Chen S.M. , et al., (1994) Am J Trop Med Hyg 50: 52-58 Dawson J.E. et al., (1992) Am J Vet Res 53: 1322-1327. Dawson J.E. et al., (1991) J Infect Dis 163: 564-567. Donatien, et al., (1935) Bull Soc Pathol Exot 28: 418-9. Ewing, (1935) J Am Vet Med Assoc 143: 503-6. Groves M.G., et al., (1975) Am J Vet Res 36: 937-940. Harrus S., et al., (1998) J Clin microbiol 36: 73-76. Jameson B.A., et al., (1988) CABIOS 4: 181-186. Jongejan F., et al., (1993) Rev Elev Med Vet Pays Trop 46: 145-152. McBride J. W., et al., (1996) J Vet Diag Invest 8: 441-447 McBride, et al.,. (1999) Clin Diagn Lab Immunol .; (In I press). ,! McClure, (1985) Ann Rev Biochem 54: 171-204. 'McGeoch D.J. (1985) Virus Res 3: 271-286. Nyindo, M., et al., (1991) Am J Vet Res 52: 1225-1230. I Nyindo, et al., (1971) Am J Vet Res 32: 1651-58.

Dawson J.E. et al., (1992) Infect Immun 66: 132-9. Dawson J.E. et al., (1992) J Clin Microb 36: 2671.80. I Reddy, et al., (1998) Biochem Biophys Res Comm 247: 636-43. i Rikihisa, et al., (1994) J Clin Microbiol 32: 2107-12. i Rothbard J.B. et al., (1988) The EMBO J7: 93-100. Sambrook J., et al., (1989) In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Press. Troy G. C. et al., (1990) Canine ehrlichiosis. Infectious I diseases of the dog and cat. Green C. E. (ed). Philidelphia: W. B. Sauders Co. von Heijne, (1986) Nucí Acids Res 14: 4683-90. | i Walker, et al., (1970) J Am Vet Med Assoc 157: 43-55.; Weiss E., et al., (1975) Appl Microbiol 30: 456-463. Yu, et al., (1997) Gene 184: 149-154. Yu, et al., (1998) J. Clin. Microbiol. (In press). Any patent or publications mentioned in this specification are indicative of the standards of those skilled in the art to which the invention belongs. These patents and publications are incorporated herein by reference by the same scope as the individual publication was individually indicated to be incorporated by reference. - u-Htoa ,, ..- .. ~ j * .. - * a *? AA *. A person skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives ii and obtain the purposes and advantages mentioned, I as well as those that are inherent to them. The present examples together with the methods, procedures, I treatments, molecules, and specific compounds described herein are representative of the preferred embodiments at present, are examples, and are not intended to limit the scope of the invention. The changes thereof and other uses I will be presented by those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. ^^ * ^^ ij¿yya »g? LIST OF SEQUENCES < 110 > Walker, David K. McBride, Jere W. Yu, Xue-Jie < 120 > Genes of the Immunodominant Protein of 28-kilodalton homologous of Ehriichia canis and uses thereof < 130 > D6152PCT < 141 > 1999-1 1-30 < 150 > 09/261, 358 < 151 > 1999-03-03 < 160 > 33 < 210 > 1 < 211 > 1607 < 212 > DNA < 213 > Ehriichia canis < 220 > < 223 > nucleic acid sequence of? Ca28-l < 400 > 1 attttattta ttaccaatct tatataatat attaaatttc tc tacaaaa 50 atctctaatg ttttatacct aatatatata ttctggcttg tatetaettt 100 | gcacttccac tattgttaat ttattttcac tattttaggt gtaatatgaa 150 attcttataa ttgcaaaaaa caactgeatt aatatcatta atgtactcta 200 ttccaagcat atctttttct gatactatac aagatggtaa catgggtggt, 250 ttagtggaaa aacttctata gtatgtacca agtgtctcac attttggtag 1300 cttctcagct aaagaagaaa gcaaatcaac tgttggagtt tttggattaa, 350 aacatgattg ggatggaagt ccaatactta agaataaaca cgctgacttt '400 actgttccaa actattcgtt cagataegag aacaatecat ttctagggtt! 450 tgcaggagct atcggttact caatgggtgg eccaagaata gaattcgaaa i500 tatcttatga ageattegac gtaaaaagtc ctaatat'caa ttatcaaaat 550 gacgcgcaca ggtactgcgc tetatetcat cacacatcgg cagccatgga 600 agetgataaa tttgtcttct taaaaaacga agggttaatt gacatatcac 650 ttgcaataaa tgcatgttat gatataataa atgacaaagt acctgtttct, 700 cettatatat gcgcaggtat tggtactgat ttgatttcta tgtttgaagc 750 tacaagtcct aaaatttcct accaaggaaa actgggcatt agttacteta 800 | ttaatccgga aacctctgtt ttcatcggtg ggcatttcca caggatcata 850 ggtaatgagt ttagagatat tcctgcaata gtacctagta actcaactac 900 aataagtgga ccacaatttg caacagtaac actaaatgtg tgtcactttg ¡950 * - * --- • »^ gtttagaact tggaggaaga átUjfaj tt aacttct aattttactg ttgccacata llOOD ttaaaaatga tctaaacttg tttztawtat tgctacatac aaaaaaagaa the bear aaatagtggc aaaagaatgt agcaataaga gggggggggg ggaccaaatt iioo tatcttctat gcttcccaag ttt tcycg ctatttatga cttaaacaac 1150 agaaggtaat atcctcacgg aaaacttatc ttcaaatatt ttatttatta i oo ccaatcttat ataatatatt ttctct aaa! tacaaaaatc actagtattt 1250 tataccaaaa tatatattct gacrtgcttt tcttctgcac ttctactatt 1300 tttaatttat ttgtcactat taggttataa taawatgaat tgcmaaagat 1350! ttttcatagc aagtgcattg atatcactaa tgtctttctt acctagcgta 1400 tctttttctg aatcaataca tgaagataat ataaatggta acttttacat 1450 tagtgcaaag gtgcctcaca tatatgccaa ttttcagtta ctttggcgta aaacacaaca i500 i550 aagaagagaa actggagttt tcggattaaa acaagattgg gacggagcaa cactaaagga tgcaagcwgc agccacacaw tagacccaag tacaatg i600 i607 < 210 > 2 < 211 > 278 < 212 > PRT < 213 > Ehriichia canis < 220 > I < 223 > sequence of the amino acids of the ECa28-1 protein < 400 > I Met Asn Cys Lys Lys lie Leu lie Thr Thr Ala Leu lie SerLeu Met Tyr Ser lie Gly Asn Met Gly Ser Val Ser His 50 55 60 Ser Thr Val Gly Val Phe Gly Leu Lys His Asp Trp Asp Gly, Ser 65 70! 75 Pro lie Leu Lys Asn Lys His Wing Asp Phe Thr Val Pro Asn¡ Tyr 80 85 90 Ser Phe Arg Tyr Glu Asn Asn Pro Phe Leu Gly Phe Wing Gly¡ Wing 95 100 105 lie Gly Tyr Ser Met Gly Gly Pro Arg He Glu Phe Glu He Ser 110 115 120 Tyr Glu Wing Phe Asp Val Lys Ser Pro Asn He Asn Tyr Gln Asp 125 130 135 Asp Ala His Arg Tyr Cys Ala Leu Ser Kis His Thr Ser Ala Ala 140 145 150 Met Glu Wing Asp Lys Phe Val Phe Leu Lys Asn Glu Gly Leu 155 155 165 Asp He Ser Leu Wing He Asn Wing Cys Tyr Asp He He Asn Asp 170 175 180 Lys Val Pro Val Ser Pro Tyr He Cys Ala Gly He Gly Thr Asp 185 190 195 Leu He Ser Met Phe Glu Wing Thr Ser Pro Lys He Ser Tyr Gln 200 205 210 Gly Lys Leu Gly He Ser Tyr Ser He Asn Pro Glu Thr Ser Val 215 220 225 i Phe He Gly Gly His Phe His Arg He He Gly Asn Glu Phe Are 230 235 240 Asp He Pro Ala He Val Pro Ser Asn Ser Thr Thr He Ser Gly 245 250 255 i Pro Gln Phe Ala Thr Val Thr Leu Asn Val Cys His Phe Gly Leu 260 265 270 Glu Leu Gly Gly Arg Phe Asn Phe 275 < 210 > 3 < 211 > 849 < 212 > DNA < 213 > Ehriichia canis < 220 > < 221 > mat_peptide < 223 > nucleic acid sequence of ECa28SA2 < 400 > 3 atgaattgta aaaaagtttt cacaataagt gcattgatat catecatata! 50 cttcctacct aatgtctcat actctaaccc agtatatggt aacagtatgt 100 atggtaattt ttacatatca ggaaagtaca tgccaagtgt tcctcatttt 150 ggaatttttt cagctgaaga agagaaaaaa aagacaactg tagtatatgg 200 cttaaaagaa aactgggcag gagatgcaat ate agtcaa agtccagatg 250 ataattttac cattcgaaat tacteattea agtatgcaag caacaagttt | 300"-" ----- • ttagggtttg cagtagctat tggttactcg ataggcagtc aga caagaa 350 agttgagatg tcttatgaag catttgatgt gaaaaatcca ggtgataatt 400 acaaaaacgg tgcttacagg tattgtgc t tatctcatca agatgatgcg 45 gatgatgaca tgactagtgc aactgacaaa tttgtatatt taattaatga 50 aggattactt aacatatcat ttatgacaaa catatgttat gaaacagcaa 55 gcaaaaatat acctctctct ccttacatat gtgcaggtat tggtac gat 60 ttaattcaca tgtttgaaac tacacatcct aaaatttctt atcaaggaaa 65 gctagggttg gcctacttcg taagtgcaga gtcttcggtt tcttttggta 70 tatattttca taaaattata aataataagt ttaaaaatgt tccagccatg 75.0 gtacctatta actcagacga gatagtagga ccacagtttg caacagtaac 8QO i attaaatgta tgctactttg gattagaact tggatgtagg ttcaacttc 849 < 210 > 4 < 211 > 283 < 212 > PRT < 213 > Ehriichia canis < 220 > < 223 > amino acid sequence of the ECa28SA2 protein < 400 > 4 Met Asn Cys Lys Lys Val Phe Thr He Ser Wing Leu He Ser Ser 5 10 15 He Tyr Phe Leu Pro Asn Val Ser Tyr Ser Asn Pro Val Tyr Gly 20 25 30 Asn Ser Met Tyr Gly Asn Phe Tyr He Ser Gly Lys Tyr Met Pro 35 40 45 Ser Val Pro His Phe Gly He Phe Ser Ala Glu Glu Glu Lys Lys 50 55 60 Lys Thr Thr Val Val Tyr Gly Leu Lys Glu Asn Trp Wing Gly Asp 65 70 75 Wing I Be Ser Gln Ser Pro Asp Asp Asn Phe Thr He Arg Asn 80 85 90 Tyr Ser Phe Lys Ala He Gly Tyr 110 115 120 Ser Tyr Glu "Wing" Phe Asp Val Lys Asn Pro Gly Asp Asn Tyr ^ ys 125 130 ¡135 - ~ ** ~ * ~ to sn Gly Wing Tyr Arg Tyr Cys Wing Leu Ser His Gln Asp Asp Wing 14C .45 150 Asp Asp Asp Met Thr Ser Wing Thr Asp Lys Phe Val -Tyr Leu He 155 160 1165! Asn Glu Gly Leu Leu Asn He Be Phe Met Thr Asn He Cys iTyr 170 175 180 Glu Thr Ala Ser Lys Asn He Pro Leu Ser Pro Tyr He Cys Wing 185 190 195 Gly He Gly Thr Asp Leu He His Met Phe Glu Thr Thr His Pro 200 205 210 I Lys He Ser Tyr Gln Gly Lys Leu Gly Leu Ala Tyr Phe Val (Ser 215 220 225 Wing Glu Ser Ser Val Ser Phe Gly He Tyr Phe His Lys He He 230 235 | 240 Asn Asn Lys Phe Lys Asn Val Pro Wing Met Val Pro He Asnj Ser 245 250 | 255 Asp Glu He Val Gly Pro Gln Phe Ala Thr Val Thr Leu Asn¡ Val 260 265 '270 Cys Tyr Phe Gly Leu Glu Leu Gly Cys Arg Phe Asn Phe 275 280 ' < 210 > 5 < 211 > 840, < 212 > DNA < 213 > Ehriichia cernís < 220 > < 221 > mat_peptide < 223 > nucleic acid sequence of ECa28SA3 < 400 > May 1 atgaattgca aaaaaattct tataacaact gcattaatgt cattaatgta 50 ctatgctcca agcatatctt tttctgatac tatacaagac gataacactg 100 catcagtgga gtagcttcta aaatatgtac caagtgtttc acattttggt 150 gttttctcag aagaaactca ctaaagaaga actgttggag tttttggatt |! 200 aaaacatgat tggaatggag gtacaatatc taactcttct ccagaaaata 250 tattcacagt tcaaaattat tcgtttaaat acgaaaacaa cccattctta 300 gggtttgcag gagctattgg ttattcaatg ggtggcccaa gaatagaact 350 tgaag cg tacgagacat tcgatgtgaa aaatcagaac aataattata 400 J ^^ • "* - línr t - '-Mjáa-fa-aat-aÉ - ...- ^ i-ii ttijM agaacggcgc acacagatac tgtgctttat ctcatcatag t cagcaaca 450 agcatgtcct ccgcaagtaa caaatttgtt ttcttaaaaa a gaagggtt: 00 aattgactta tcatttatga taaatgcatg ctatgacata ataattgaag 5¡50 gaatgccttt ttcaccttat at GTGCAG gtgttggtac tgatgttgtt 6.00 tccatgtttg aagctataaa tcctaaaatt tcttaccaag gaaaactag 650 attaggttat agtataagtt cagaagcctc tgtttttatc ggtggacact 100 ttcacagagt cataggtaat gaatttagag acatccctgc tatggttcct 750 atcttccaga agtggatcaa aaaccaattt gcaatagtaa cactaaatg 800 840 gtgtcacttt ggcatagaac ttggaggaag atttaacttc < 210 > 6 < 211 > 280 < 212 > PRT < 213 > Ehriichia canie < 220 > < 223 > sequence of the amino acids of the ECa28SA3 protein < 400 > 6 I Met Asn Cys Lys Lys He Leu He Thr Thr Wing Leu Met Ser Leu 5 10 | 15 Met Tyr Tyr Ala Pro Ser He Phe Ser Asp Thr He GlnjAsp 20 25 i 30 Asp Asn Thr Gly Ser Phe Tyr He Ser Gly Lys Tyr Val Pro Ser 35 40 45 Val Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Arg Asnj Ser 50 55 '60 Thr Val Gly Val Phe Gly Leu Lys His Asp Trp Asn Gly Gly, Thr 65 70! 75 I have been Asn Being Ser Glu Asn He Phe Thr Val Gln Asn¡ Tyr 80 85 ¡90 Being Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Wing Gly¡ Wing 95 100! 105 He Gly Tyr Ser Met Gly Gly Pro Arg He Glu Leu Glu Vali Leu 110 115 120 Tyr Glu Thr Phe Asp Val Lys Asn Gln Asn Asn Asn Tyr Lys Asn 125 130 135 Gly Ala His Arg Tyr Cys Ala Leu Ser His His Ser Ser Ala Thr 140 145 150 Being Met Being Being Wing Being Asn Lvs Phe Val Phe Leu Lvs Asn | Glu 155 160 165 Gly Leu He Asp Leu Being Phe Met He Asn Aia Cys Tyr Asp He 170 175 180 He He Glu Gly Met Pro Phe Ser Pro Tyr He Cys Ala Gly Val 185 190 195 Gly Thr Asp Val Val Ser Met Phe Glu Ala He Asn Pro Lys He 200 205 210 Ser Tyr Gln Gly Lys Leu Gly Leu Gly Tyr Ser He Ser Ser Glu 215 220 225 Wing Ser Val Phe He Gly Gly His Phe His Arg Val He Gly Asn 230 235 240 Glu Phe Arg Asp He Pro Wing Met Val Pro Ser Gly Ser Asn Leu 245 250 255 Pro Glu Asn Gln Phe Wing He Val Thr Leu Asn Val Cys His Phe 260 265 270 Gly He Glu Leu Gly Gly Arg Phe Asn Phe 275 280 < 210 > 7 < 211 > 133 < 212 > PRT < 213 > Ehriichia canis < 220 > < 223 > partial sequence of the amino acid of the ECa2ßSA2 protein < 400 > 7 Met Asn Cys Lys Lys Val Phe Thr He Ser Wing Leu He Serj Ser 5 10 15 He Tyr Phe Leu Pro Asn Val Ser Tyr Ser Asn Pro Val Tyr¡ Gly 20 25 '; 30 Asn Ser Met Tyr Gly Asn Phe Tyr He Ser Gly Lys Tyr Met; Pro 35 40 45 Ser Val Pro His Phe Gly He Phe Ser Ala Glu Glu Glu Lys Lys 50 55! 60 Lys Thr Thr Val Val Tyr Gly Leu Lys Glu Asn Trp Wing Gly Asp 65 70 Wing 75 Wing Be Ser Gln Ser Pro Asp Asp Asn Phe Thr He Arg Asn 80 85! 90"- • -" • "- • -» - * - < "- < * - i ~ -f *? ia ** m? * L? iA? I Tyr Ser Phe Lys Tyr Wing Ser Asn Lys Phe Leu Gly Phe Wing Val 95 100 105 Wing He Giy Tyr Ser He Gly Ser Pro Arg He Glu Val Glu Met 110 115 120 Ser Tyr Glu Ala Phe Asp Val Lys Asn Gln Gly Asn Asn 125 130 < 210 > 8 < 211 > 287 ¡< 212 > PRT < 213 > Ehriichia canis < 220 > < 223 > amino acid sequence of the ECa28SA1 protein i < 40C > 8 Met Lys Tyr Lys Lys Thr Phe Thr Val Thr Wing Leu Val Leu1 Leu 5 10 '15 Thr Ser Phe Thr His Phe He Pro Pro Phe Tyr Ser Pro Ala Arg Ale 20 25, 30 Ser Thr He His Asn Phe Tyr He Ser Gly Lys Tyr Met Pro1 Thr 35 40 45 Wing Ser His Phe Gly He Phe Ser Wing Lys Glu Glu Gln Serj Phe 50 55, 60 Thr Lys Val Leu Val Gly Leu Asp Gln Arg Leu Ser His Asn | He 65 70 | 75 He Asn Asn Asn Asp Thr Ala Lys Ser Leu Lys Val Gln Asri Tyr 80 85 '90 Ser Phe Lys Tyr Lys Asn Asn Pro Phe Leu Gly Phe Wing Gly Wing 95 100 105 He Gly Tyr Being He Gly Asn Being Arg He Glu Leu Glu Val Ser 110 115 120 His Glu He Phe Asp Thr Lys Asn Pro Gly Asn Asn Tyr Leu Asn 125 130 135 Asp Ser His Lys Tyr Cys Ala Leu Ser His Gly Ser His He Cys 140 145 150 Ser Asp Gly Asn Ser Gly Asp Trp Tyr Thr Wing Lys Thr Asp Lys 155 160 165 Phe Val Leu Leu Lys Asn Glu Gly Leu Leu Asp Val Ser Phe Met 170 175 180 - '- "- - * - g & Leu Asn Ala Cys Tyr Asp He Thr Thr Glu Lys Met Pro Phe Ser 185 190 L95 Pro Tyr He Cys Wing Gly He Gly Thr Asp Leu He Ser Met Phe 200 205 210 I Glu Thr Thr Gln Asn Lys He Ser Tyr Gln Giy Lys Leu Gly Leu 215 220 225 Asn Tyr Thr He Asn Ser Arg Val Ser Val Phe Aia Gly Gly His 230 235 240 Phe His Lys Val He Gly Asn Glu Phe Lys Gly He Pro Thr Leu 245 250 ¡255 Leu Pro Asp Gly Ser Asn He Lys Val Gln Gln Ser Wing Thr Val 260 265 270 Thr Leu Asp Val Cys His Phe Gly Leu Glu He Gly Ser Arg | Phe 275 280! 285 Phe Phe ' < 210 > 9 < 211 > 281 < 212 > PRT < 213 > Ehriichia chaffeensis < 220 > < 223 > amino acid sequence of E. chaffeensis P28 < 400 > 9 Met Asn Tyr Lys Lys Val Phe He Thr Ser Ala Leu He Ser! eu 5 10 15 I Ser Ser Leu Pro Gly Val Ser Phe Ser Asp Pro Wing Gly, Ser 20 25 30 Gly He Asn Gly Asn Phe Tyr He Ser Gly Lys Tyr Met Pro Ser 35 40 45 Wing Ser His Phe Gly Val Phe Ser Wing Lys Glu Glu Arg Asn Thr 50 55 60 Thr Val Gly Val Phe Gly Leu Lys Gln Asn Trp Asp Gly Ser Wing 65 70 75 He Ser Asn Ser Ser Pro Asn Asp Val Phe Thr Val Ser Asn Tyr 80 85 90 Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Ala Glyj Ala 95 100 105 He Gly Tyr Ser Met Asp Gly Pro Arg He Giu Leu Glu Val Ser 110 115 120 Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys Asn 125 130 135 Glu Ala His Arg Tyr Cys Ala Leu Ser His Asn Ser Ala Aia Asp 140 145 150 Met Being Being Wing Being Asn Asn Phe Val Phe Leu Lys Asn Giu Gly 155 160 165 Leu Leu Asp He Being Phe Met Leu Asn Ala Cys Tyr Asp Val iVal 170 175 '180 Gly Glu Gly He Pro Phe Ser Pro Tyr He Cys Wing Gly He Gly 185 190 195 Thr Asp Leu Val Ser Met Phe Glu Wing Thr Asn Pro Lye He Be 200 205 '210 Tyr Gln Gly Lys Ser Val Phe He Phe Arg Asp He Pro Thr He He Pro Thr Gly Ser Thr Leu Wing 245 250 '255 Gly Lys Gly Asn Tyr Pro Wing He Val He Leu Asp Val Cys His His 260 265 ¡270 Phe Gly He Glu Leu Gly Gly Arg Phe Ala Phe '275 280 < 210 > • 10 < 211 > 283 < 212 > PRT < 213 > Ehriichia chaffeensis < 220 > < 223 > amino acid sequence d »< 400 > 10 Met Asn Tyr Lys Lys He Phe Val Ser Ser Ala Leu He Ser Leu 5 10 ¡15 Met Ser He Leu Pro Tyr Gln Be Phe Wing Asp Pro Val Thr Ser 20 25! 30 ***** * • • '- «»! - '^ - "*** - -' -" | | Asn Asp Thr Gly Val Lys Tyr Asn 50 55 ¡60 Glu Glu Ala Pro He Asn Gly Asn Thr Ser He Thr Lys Lys' Val 65 70! 75 Phe Gly Leu Lys Lys Asp Gly Asp He Wing Gln Ser Wing Asn Phe 80 85 i 90 i Asn Arg Thr Asp Pro Wing Leu Glu Phe Gln Asn Asn Leu lie Be 95 100! 105 Gly Phe Ser Gly Ser He Gly Tyr Wing Met Asp Gly Pro Arg He 110 115 120 Glu Leu Glu Wing Wing Tyr Gln Lys Phe Asp Wing Lys Asn Pro Asp 125 130 '135 Asn Asn Asp Thr Asn Ser Gly Asp Tyr Tyr Lys Tyr Phe Gly Leu 140 145 150 Ser Arg Glu Asp Wing He Wing Asp Lys Lys Tyr Val Val Leu Lys 155 160 '165 Asn Glu Gly He Thr Phe Met Ser Leu Met Val Asn Thr Cys Tyr 170 175 ii 180 Asp He Thr Ala Glu Gly Val Pro Phe He Pro Tyr Wing Cys Wing 185 190 195 Gly Val Gly Wing Asp Leu He Asn Val Phe Lys Asp Phe Asn Leu 200 205 210 Lys Phe Ser Tyr Gln Gly Lys He Gly He Ser Tyr Pro lie Thr 215 220 '225 I Pro Glu Val Ser Wing Phe He Gly Gly Tyr Tyr His Gly Val He 230 235 240 Gly Asn Asn Phe Asn Lys He Pro Val He Thr Pro Val Val Leu 245 250! 255 Glu Gly Ala Pro Gln Thr Thr Ser Ala Leu Val Thr He Asp Thr 260 265 270 Gly Tyr Phe Gly Gly Glu Val Gly Val Arg Phe Thr Phe 275 280 < 210 > 11 < 211 > 280 eleven "* - ~" "- - <I <212> PRT! <213> Ehriichia chaffeensis j <220> <223> amino acid sequence of .chaffeensie OM ^ -IC < 400> 11! Met Asn Cys Lys Met Ser Phe Leu Asp Ser Val Ser Gly Asn Phe Tyr He Ser Gly Lys Tyr Met Pro 35 40 45 Ser Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Lys Asn 50 55 60 Pro Thr Val Ala Leu Tyr Gly Leu Lys Gin Asp Trp Asn Gly Val 65 70 75 Be Ala Be Ser His Wing Asp Wing Asp Phe Asn Asn Lys Gly Tyr 80 85 90 Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Wing Gly Wing 95 100 I05 I He Gly Tyr Ser Met Gly Gly Pro Arg He Glu Phe Glu Val Ser 110 115 120 Tyr Glu Thr Phe Asp Val Lys Asn Gln Gly Gly Asn Tyr Lys Asn 125 130 135 Asp Wing His Arg Tyr Cys Wing Leu Asp Arg Lys Wing Ser Ser Thr 140 145 150 Asn Wing Thr Wing His Ser Tyr Val Leu Leu Lys Asn Glu Gly Leu 155 160 165 Leu Asp He Ser Leu Met Leu Asn Wing Cys Tyr Asp Val Val Ser 170 175 180 Glu Gly He Pro Phe Ser Pro Tyr He Cys Wing Gly Val Gly Thr 185 190 195 Asp Leu He Ser Met Phe Glu Wing He Asn Pro Lys He Ser Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Ser He Asn Pro Glu Wing Ser 215 220 225 Val Phe Val Gly Gly His Phe His Lys Val Wing Gly Asn Gli Phe 230 235 240 12 Arg Asp He Ser Thr Leu Lys Aia Phe Ala Thr Pro Ser Ser, Ala 245 250 255 Wing Thr Pro Asp Leu Wing Thr Val Thr Leu Ser Val Cys His Phe 260 265 270 Gly Val Glu Leu Giy Gly Arg Phe Asn Phe 275 280 - < 210 > 12 < 211 > 286 < 212 > PRT < 213 > Ehriichia chaffeensis < 220 > < 223 > amino acid sequence of? OMP-1D chaffeensis < 400 > 12 Met Asn Cys Glu Lys Phe Phe He Thr Thr Ala Leu Thr Leu Leu 5 10 15 Met Ser Phe Leu Pro Gly Be Ser Leu Ser Asp Pro Val Glni Asp 20 25 30 Asp Asn He Ser Gly Asn Phe Tyr He Ser Gly Lys Tyr Met Pro 35 40 45 Being Ala Ser His Phe Gly Val Phe Ser Ala Lys Glu Glu Arg¡ Asn 50 55 60 Thr Thr Val Gly Val Phe Gly He Glu Gln Asp Trp Asp Arg Cys 65 70 75 Val He Ser Arg Thr Thr Leu Ser Asp He Phe Thr Val Pro Asn 80 85 90 Tyr Ser Phe Lys Tyr Glu Asn Asn Leu Phe Ser Gly Phe Wing Gly 95 100. 105 Ala He Gly Tyr Ser Met Asp Gly Pro Arg He Glu Leu Glu? Val 110 115 120 Ser Tyr Glu Wing Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr, Lys i 125 130 I 135! Asn Glu Ala His Arg Tyr Tyr Ala Leu Ser His Leu Leu Gly Thr 140 145 150 Glu Thr Gln He Asp Gly Wing Gly Being Wing Being Val Phe Leu He 155 160 165 13 Asn Giu Gly Leu Leu Asp Lys Ser Phe Met Leu Asn Wing Cys Tyr 170 175 180 Asp Val He Ser Glu Gly He Pro Phe Ser Pro Tyr He Cys Wing 185 190 195 Gly He Gly He Asp Leu Val Ser Met Phe Glu Wing He Asn Pro 200 205 210 Lys He Ser Tyr Gln Gly Lys Leu Gly Leu Ser Tyr Pro He i Ser 215 220 225 Pro Glu Wing Ser Val Phe He Gly Gly His Phe His Lys Val He 230 235 240 Gly Asn Glu Phe Arg Asp Lie Pro Thr Met He Pro Ser Glu 'Ser 245 250 255 Ala Leu Ala Gly Lys Gly Asn Tyr Pro Ala He Val Thr Leu Asp 260 265 270 Val Phe Tyr Phe Gly He Glu Leu Gly Gly Arg Phe Asn PheiGln 275 280 285 Leu < 210 > 13! < 211 > 278 I < 212 > PRT | < 213 > Ehriichia chaffeensis, < 220 > < 223 > amino acid sequence of E. chaffeensis OMP-1E < 400 > 13! i Met Asn Cys Lys Lys Phe Phe He Thr Thr Ala Leu Val Ser! Leu 5 10 15 Met Ser Phe Leu Pro Gly He Ser Phe Ser Asp Pro Val Gln Gly 20 25 30 Asp Asn He Ser Gly Asn Phe Tyr Val Ser Gly Lys Tyr Met Pro 35 40 45 Being Ala Ser His Phe Gly Met Phe Being Ala Lys Glu Glu Lys¡ Asn 50 55 ¡60 Pro Thr Val Ala Leu Tyr Gly Leu Lys Gln Asp Trp Glu Gly He 65 70! 75 Being Being Being His As Asp Asn His Phe Asn Asn Lys Gly Tyr 80 85 90 14 _ ^ _ j¡y¡¿ ^ * faith * ¡^ ¿^ iíw ^^^ > Be Phe Lys Tyr Glu Asn Asn Pro Phe Leu Giy Phe Wing Gly Wing 95 100 105 He Gly Tyr Ser Met Gly Gly Pro Arg Val Glu Phe Glu Val Ser 110 115 120 Tyr Giu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr Lys Asn 125 130 135 Asp Wing His Arg Tyr Cys Wing Leu Gly Gln Gln Asp Asn Ser Gly 140 145 150 He Pro Lys Thr Ser Lys Tyr Val Leu Leu Lys Ser Glu Gly i Leu 155 160 165 Leu Asp I Am Being Phe Met Leu Asn Ala Cys Tyr Asp I have He Asn Glu Being He Pro Leu Being Pro Tyr He Cys Wing Gly Val Gly > Thr 185 190 195 Asp Leu He Ser Mét Phe Glu Wing Thr Asn Pro Lys He Ser Tyr 200 205 210 Gln Gly Lys Leu Gly Leu Ser Tyr Ser He Asn Pro Glu Ala i Ser 215 220 225 Val Phe He Gly Gly His Phe His Lys Val He Gly Asn Glu Phe 230 235 ¡240 Arg Asp He Pro Thr Leu Lys Ala Phe Val Thr Ser Ser Ala ¡Thr 245 250 255 Pro Asp Leu Ala He Val Thr Leu Ser Val Cys His Phe Gly He 260 265, 270 Glu Leu Gly Gly Arg Phe Asn Phe 275 < 210 > 14 < 211 > 280 < 212 > PRT < 213 > Ehriichia chaffeensis < 220 > < 223 > amino acid sequence of E. chaffeensis 0MP-1F < 400 > 14 I Met Asn Cys Lys Lys Phe Phe He Thr Thr Thr Leu Val Ser¡Leu 5 10 ¡15 fifteen Met Ser Phe Leu Pro Gly He Ser Phe Ser Asp Wing Val Gin Asn 20 25 30 Asp Asn Val Gly Asn Phe Tyr He Ser Gly Lys Tyr Val Pro 35 40 45 Ser Val Ser His Phe Gly Val Phe Ser Ala Lys Gln Glu Arg Asn 50 55 60 Thr Thr Thr Gly Val Phe Gly Leu Lys Gln Asp Trp Asp Gly Ser 65 70 75 Thr He Ser Lys Asn Ser Pro Glu Asn Thr Phe Asn Val Pro Asn 80 85 190 Tyr Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Wing Gly 95 100 105 Wing Val Gly Tyr Leu Met Asp Gly Pro Arg He Glu Leu Glu i Met 110 115 ¡120 Ser Tyr Giu Thr Phe Asp Val Lys Asn Gln Gly Asn Asn Tyr 'Lys 125 130 ¡135 Asn Asp Ala His Lys Tyr Tyr Ala Leu Thr His Asn Ser Gly¡Gly 140 145 150 Lys Leu Ser Asn Ala Gly Asp Lys Phe Val Phe Leu Lys Asn 'Glu 155 160 ¡165 Gly Leu Leu Asp He Ser Leu Met Leu Asn Ala Cys Tyr Asp Val 170 175 180 He Ser Glu Gly Gly Thr Asp Leu Ser Tyr Gln Gly 215 220 225 Wing Ser Val Phe Val Gly Gly His Phe His Lys Val He Gly Asn 230 235 240 Glu Phe Arg Asp He Pro Wing Met He Pro Ser Thr Ser Thr Leu 245 250 255 Thr Gly Asn His Phe Thr He Val Thr Leu Ser Val Cys His Phe 260 265 270 Gly Val Glu Leu Gly Gly Arg Phe Asn Phe 275 280 16 < 210 > 15 < 211 > 284 < 212 > PRT < 213 > Cowdria ruminantium < 220 > | < 223 > amino acid sequence of C. rupin ti um MAP-i < 400 > 15 Met Asn Cys Lys Lys He Phe He Thr Ser Thr Leu He Ser Leu 5 10 ¡15 Val Ser Phe Leu Pro Gly Val Ser Phe Ser Asp Val He Gln¡Glu 20 25 ¡30 Glu Asn Asn Pro Val Gly Ser Val Tyr I Have To Be Wing Lys Tyr Met 35 40 45 Pro Thr Wing His Phe Gly Lys Met Ser He Lys Glu Asp Ser 50 55 60 Arg Asp Thr Lys Wing Val Phe Gly Leu Lys Lys Asp Trp Asp 'Gly 65 70 75 Val Lys Thr Pro Ser Gly Asn Thr Asn Ser He Phe Thr Glu i Lys 80 85 90 Asp Tyr Ser Phe Lys Tyr Glu Asn Asn Pro Phe Leu Gly Phe Wing 95 100 105 Gly Wing Val Gly Tyr Ser Met Asn Gly Pro Arg He Glu Phe ! Glu 110 115 120 Val Ser Tyr Glu Thr Phe Asp Val Arg Asn Pro Gly Gly Asn Tyr 125 130 135 Lys Asn Asp Ala His Met Tyr Cys Ala Leu Asp Thr Ala Ser Ser 140 145 150 Ser Thr Ala Gly Ala Thr Thr Ser Val Met Val Lys Asn Glu Asn 155 160 165 Leu Thr Asp Be Ser Leu Met Leu Asn Wing Cys Tyr Asp He Met 170 175 180 Leu Asp Gly Met Pro Val Ser Pro Tyr Val Cys Ala Gly He Gly 185 190 195 Thr Asp Leu Val Ser Val He Asn Ala Thr Asn Pro Lys Leu Ser 200 205 210 Tyr Gln Gly Lys Leu Gly He Ser Tyr Ser He Asn Prt > Glu Ala 215 220 225 17 - - "•• * ~ p ** • ** .- .., .... ^ .é .. ...,." "T .., .... < M" «* ^ * M. ^ -MißH ^ ktfM Ser He Phe He Gly Gly Kis Phe Kis Arg Val He Gly Asri Glu 230 235 240 Phe Lys Asp He Wing Thr Ser Lys Val Phe Thr Ser Ser Gly Asn 245 250 255 Wing Being Ser Wing Val Ser Pro Gly Phe Wing Being Wing He Leu Asp 260 265 270 Val Cys His Phe Gly He Glu He Gly Gly Arg Phe Val Phé 275 280 < 210 > 16 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > < 222 > linker 313-332 of c. ruzpinantium MAP-1, also nucleotides 307-326 of E. chaffee? sie P28 < 223 > 793 front primers for PCR < 400 > 16 gcaggagctg ttggttactc 20 < 210 > 17 < 211 > 21 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > < 222 > nucleotides 823-843 of C. rt ipaptium MAP-1, also nucleotides 814-834 of E. chaffeensis P28 < 223 > reverse primer 1330 for PCR < 400 > 17 ccttcctcca agttctatgc c 21 < 210 > 18 < 211 > 24 < 212 > DNA 18 < ? 13 > artificial sequence < 220 > < 221 > link_strainer < 223 > 46f primer, specific for the gene? Ca28SA2 < 400 > 18 atatacttcc tacctaatgt ctca 24 < 210 > 19 < 211 > 20 < 212 > DNA < 213 > < 220 > < 221 > starter_link, < 223 > primer used to sequence the genes of the 28-kDa protein in E. canis ¡< 400 > 19! agtgcagagt cttcggtttc 20 < 210 > 20 < 211 > 18 < 212 > DNA < 213 > < 220 > < 221 > link_strainer < 223 > primer used to sequence the 28-kDa protein genes in E. canis < 400 > twenty gttacttgcg gaggacat 18 < 210 > 21 < 211 > 24 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > starter_band < 222 > nucleotides 6 6887-710 of ECa28-l 19 < 223 > 394 primer for PCR < 400 > twenty-one gcatttccac aggatcatag gtaa 24 < 210 > 22 < 211 > 24 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > band_highlight < 222 > nucleotides 710-687 of ECa28-l < 223 > 394C primer for PCR < 400 > 22 ttacctatga tcctgtggaa atgc 24 < 210 > 23 < 211 > 20 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > starter_link < 223 > r c > < e-hbaardtro? rr 77Q93 m hybridizes to a region with i Eca28-1 used to amplify the intergenic region between the gene ECa28SA3 and ECa28-1. < 400 > 2. 3 gagtaaccaa cage tec tgc 20 < 210 > 24 < 211 > 24 < 212 > DNA < 213 > artificial sequence < 220 > < 221 > flyer_strider twenty from? Ca28-l tctactttgc acttccacta ttgt 24 < 210 > 25 < 212 > DNA < 220 > < 221 > Cebador_banda Cß &ampññ EC250W-! encoders adjacent to the open reading structure of ^ < nns attcttttgc cactattttt cttt 24 < 210 > 26 < 212 > DNA ¡-i a-, < r22Q > '< 221 > Primer_link = ¿23? Primer ECaSAS -2 corresponded to Iss regions depíre of ECa28SA3, used to amplify the iptergenic region NC3 < 4üü 2 & ct ggattag gctatagtat aagtt 25 < 210 > 27 ¡211 * 23 i < 212 > PRT < Z2.3 = > Jif chiu c ms < 220 > ! < 221 > PEPTIDO --223 ups signal fFFsrrpipat predicts the peptide of ECa2S-1 i twenty-one and? Ca23SA3 < 4CC > 27 Met Asn Cys Lys Lys He Leu He Thr Thr Aia Leu Ket Ser Leu 5 1C, 15 Met Tyr Tyr Ala Pro Ser He Ser 20 < 210 > - 28 < 211 > 25 < 212 > PRT < 213 > Ehriichia chaffeensis < 220 > < 223 > Amino acid sequence of: E. chaffeensis P28 < 400 > 28 Met Asn Tyr Lys Lys lie Leu He Thr Ser Wing Leu He Ser Leu 5 10 15 He Ser Ser Leu Pro Gly Val Ser Phe Ser 20 25 < 210 > 29 < 211 > 26 < 212 > PRT < 213 > Ehriichia canis < 220 > < 223 > Amino acid sequence of the putative cleavage site of ECa28-1 < 400 > 29 Met Asn Cys Lys Lys He Leu lie Thr Thr Ala Leu He Ser Leu 5 10! 15 Met Tyr Ser He Pro Ser He Ser Ser Phe Ser 20 25 i < 210 > 30 < 211 > 299 ~ n _JMÍi¡_ _M- * i-Í ---- l *. . ,. . -j ^ jt-Jw- < 212 > DNA < 213 > ? hriichia canie < 220 > < 222 > Nucleic acid sequence of intergenic non-coding region 1 (28NC1) < 400 > 30 taatacttct attgtacatc ttaaaaatas tactastttg cttctstsg '50 aagagagaaa ttataaacgc tagttagtaa taaattagaa ag aaatat 100 tagaaaagtc atatgttttt cattgtcatt gatactcaac taaaagtagt 150 crtattaata ataaatgtta gtatattaaa attttacgta tttcccttac 200 aaaagccact agtattttat actaaaagct atactttggc ttgtatttaa 250 tttgtatttt tactactg t aatttacttt cacrgtttcr ggtgtaaa- 299 < 210 > 31 < 2ii > 345 < 212 > DNA < 213 > Ehriichia canis < 220 > < 223 > Nuc intergenic acid sequence (28NC2) < 400 > 31 taatttcgtg gtacacatat cacgaagcta aaattgtttt tttatctctg 50 ctgtatacaa gagaaaaaat agtagtgaaa attaectaac aatatgacag 100 tacaagttta ecaagettat tctcacaaaa cttcttgtgt cttttatctc 150 tttacaatga aatgtacact tagetteact actgtagagt gtgtttatca 2¡00 atgctttgtt tattaatact ctacataata tgttaaattt ttcttacaaa 250 acteactagt aatttatact agaatatata ttctgacttg tatttgcttt 300 atacttccac tattgttaat ttattttcac tattttaggt gtaat 3 ^ 5 < 210 > 32 < 211 > 345 * < 212 > < 213 > Ehriichia canis < 220 > DNA < 223 > Sequence of intergenic acid (28NC3) < 400 > 32 2. 3 ! egatettatt gttgccacat taaaaatg atctaaactt gtttttatta 50 ttgctacata caaaaaaaag aaaaatagtg gcaaaagaat gtagcaataa 100! gagggggggg ggggactaaa tttaccttct attcttctaa tattctttac 150 tatattcaaa tagcacaact caatgcttcc aggaaaatat gtttctaata 20C i ttttatttat taccaatcct tataaatat attaaatttc tcttacaaaa 250 atctctaatg ttttatactt aa ataata ttctggc tg tatttacttt 300i gcacttccac tattgttaat ttattttcac tattttaggt gtaat 345 ' < 210 > 33 < 211 > 355 < 212 > DNA < 213 > Ehriichia canis < 220 > < 223 > Nucleic acid sequence of integnetic non-coding region 4 (28NC4) < 400 > 33 taattttatt gttgccacat attaaaaatg atctaaactt gtttttawta 50 caaaaaaaga ttgctacata aaaatagtgg caaaagaatg tagcaataag 100 aggggggggg gggaccaaat ttatcttcta tgcttcccaa gttttttcyc 150 acttaaacaa gctatttatg cagaaggtaa tatcctcacg gaaaacttat 200 cttcaaatat tttatttatt accaatctta tataatatat taaatttctc 250 ttacaaaaat cactagtatt ttataccaaa atatatattc tgacttgctt 300 ttcttctgca cttctactat ttttaattta tttgtcacta ttaggttata taaw 350 355 24 .e $ ¿«- ** &B4

Claims (19)

CLAIMS j Having described the invention as above, I claim as property what is contained in the following! claims!

1. The DNA sequences that encode a 30-kilodalton protein of Ehriichia canis, characterized in that said protein is immunoreactive with antiserum Ehriichia canis, and wherein said protein contains an amino acid sequence selected from the group consisting of SEQ ID No. 4 and SEQ ID No. 6.! I

2. The DNA sequences of claim 1, characterized in that said protein has an N-terminal signal sequence.

3. The DNA sequences of claim 1, characterized in that said protein is post-translationally modified to a 28-kilodalton protein.

4. The DNA sequences of claim 1, characterized in that the DNA has a sequence 1 selected from the group consisting of SEQ ID No. 3? and SEQ ID No. 5.

5. A vector comprising the DNA sequences I of claim 1, characterized in that said A N is contained in a single place or locus of Ehriichia canis.

6. The DNA sequences of claim 5, characterized in that said site or locus is a locus or locus of multiple genes of 5,592 kb in length.

7. The DNA sequences of claim 6, characterized in that said site or locus encodes 28-kilodalton homologous proteins of Ehriichia canis.

8. The DNA sequences of claim 7, characterized in that said homologous 28-kilodalton proteins of Ehriichia canis are selected from the group consisting of Eca28SAl, Eca28SA2, Eca28SA3, Eca28-1 and Eca28-2.

9. A vector comprising the DNA sequences of claim 1.

10. The vector of claim 9, characterized in that said vector is an expression vector capable of expressing a peptide or polypeptide encoded by the sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 and SEQ ID NO: 3. NO: 5, when said expression veptor is introduced into a cell.

11. A characterized recombinant protein, because it comprises the amino acid sequence is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

12. The recombinant protein of claim 12, characterized in that said amino acid sequence is encoded by a nucleic acid segment comprising a sequence selected from the group consisting of SEQ. NO: l, SEQ ID NO: 3 and SEQ ID NO: 5.

13. A host cell, characterized in that it comprises the nucleic acid segment selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5.

14. A method for producing the recombinant proteote of claim 11, characterized in that it comprises the steps of: obtaining a vector comprising an expression region comprising a sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NO. NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 operatively linked to a promoter; transfecting said vector into a cell; and culturing said cells under conditions effective for the expression of said expression region.

15. An immunoreactive antibody with an amino acid sequence, characterized in that it is selected from the group consisting of SEQ ID NO: and SEQ ID NO: 6.

16. A method for inhibiting the infection of Ehrlichia canis in a subject, characterized in that it comprises the steps of: identifying a subject suspected of being exposed to or infected with Ehriichia canis; and administering a composition comprising a 28-kDa antigen of Ehriichia canis in an amount I effective to inhibit an infection of Ehriichia canis.

17. The method of claim 16, wherein said 28 kDa antigen is a recombinant protein characterized in that it comprises an amino acid sequence 1 selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.

! 18. The method of claim 17, characterized in that said recombinant protein is encoded by a gene comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. !

19. The method of claim 17, characterized in that said recombinant protein is dispersed in a pharmaceutically acceptable carrier.