Isolated dna sequence encoding ehrlichia canis 30 kilodalton protein, vector, recombinant protein and method for its preparing, cell-host, antibody and method for inhibition of ehrlichia canis infection in subject

FIELD: molecular biology, veterinary.

SUBSTANCE: invention proposes isolated DNA sequence (variants) encoding Ehrlichia canis protein of size 30 kDa. Also, invention proposes vector comprising such sequence, recombinant Ehrlichia canis 28 kDa protein encoded by this sequence, a cell-host comprising this sequence, a method for preparing the protein, immunoreactive antibody specific to this protein and a method for inhibition of Ehrlichia canis infection in subject. Recombinant protein of size 28 kDa from Ehrlichia canis shows immune reactivity with respect to serum against Ehrlichia canis. Proposed group of inventions can be used in development of vaccines and serodiagnosticum that shows high effectiveness for prophylaxis of diseases and for carrying out the serodiagnosis.

EFFECT: improved preparing method, valuable medicinal and veterinary properties of protein.

19 cl, 17 dwg, 8 ex

 

The technical field of the invention

This invention relates primarily to the field of molecular biology. More specifically, this invention relates to the molecular cloning and characterization of genes homologous proteins of Ehrlichia canis 28-kDa multigene locus, codereuse homologous proteins of Ehrlichia canis 28-kDa, and their application.

Description related field

Arlais dogs, also known as tropical premieropinion dogs, represents the wrapping tongs riccadonna disease dogs, first described in Africa in 1935 and the United States in 1963 (Donatien and Lestoquard, 1935; Ewing, 1963). The disease became better recognized after his epizootic outbreak that occurred among U.S. military dogs during the Vietnam war (Walker et al., 1970).

Etiological agent erlichiosis dogs is Ehrlichia canis, a small gram-negative, obligate intracellular bacterium that exhibits tropism in respect of mononuclear phagocytes (Nyindo et al., 1971) and transferred tick brown dog, Rhipicephalus sanguineus (Groves et al., 1975). With the development of erlichiosis dogs may be a manifestation of the three forms - acute, subclinical and chronic. The acute form is characterized by fever, anorexia, depression, lymphadenopathy and small thrombocytopenia (Troy and Forrester, 1990). Usually after acute is army dogs recover, but remain persistent infected carriers of the pathogen without clinical signs of disease for months and even years (Harrus et al., 1998). In some cases develop chronic form, which is manifested by thrombocytopenia, hyperglobulinemia, anorexia, exhaustion and bleeding, in particular nose bleeding, followed by death (Troy and Forrester, 1990).

Regulation of surface antigenicity may be an important factor in the mechanism of the establishment of such persistent infections in the host. Although the pathogenesis of the disease are not yet known, multigene families, described the representatives of related genera Ehrlichia, Anasplasma and Cowadria may be involved in the variability of expression of the main surface antigen that helps, therefore, to escape immune control. Anaplasma marginale, the bacterium closely related to E. canis, shows the variability of the genes of the main surface protein-3 (msp-3), which leads to antigenic polymorphism among strains (Alleman et al., 1997).

Molecular taxonomic analysis based on the gene 16S rRNA showed that E. canis and E.chaffeensis, the etiological agents of macrophage erlichiosis person (NME)are closely related (Anderson et al., 1991; Anderson et al., 1992; Dawson et al., 1991; Chen et al., 1994). Described significant cross reactivity of antigens 64, 47, 40, 30, 29, and 23-kDa between E. canis and E.chffeensis (Chen et al., 1994; Chen et al., 1997; Rikihisa et al., 1994; Rikihisa et al., 1992). Analysis of immunoreactive antigens by immunoblot using human serum and dogs taken in the recovery period, allowed us to identify many of the immunodominant proteins of E. canis, including protein 30 kDa (Chen et al., 1997). In addition, the protein of E. canis in the 30 kDa was described as a major immunodominant antigen recognized early in an immune response, which is characterized by antigenic properties of the protein from E. chaffeensis in the 30-kDa (Rikihisa et al., 1992; Rikihisa et al., 1994). Also identified other immunodominant proteins of E. canis with molecular masses ranging from 20 to 30 kDa (Brouqui et al., 1992; Nyindo et al., 1991; Chen et al., 1994; Chen et al., 1997).

Homologous immunodominant proteins 28-32-kDa encoded by multigene families, have been described in related microorganisms, including E. chaffeensis and Cowdria ruminantium (Sulsona et al., 1999; Ohashi et al., 1998a; Reddy et al., 1998). Have recently been described properties 21-membered multigene families encoding proteins of E. chaffeensis in 23-28-kDa (Yu et al., 2000). Proteins of the outer membrane E.chaffeensis 28-kDa exposed on the surface and contain three main hypervariable region (Ohashi et al., 1998a). It turned out that recombinant P28 E.chaffeensis provides protection against introduced appropriate infection in mice, and anticigarette raised against a recombinant protein that cross-reacts with the be the com E.canis 30-kDa (Ohashi et al., 1998a). Describes the diversity of the P28 gene among isolates E.chaffeensis (Yu et al., 1999a), and studies using monoclonal antibodies, in addition, revealed the diversity in downregulation of P28 proteins (Yu et al., 1993). On the contrary, describes the entire conservative P28 genes in geographically different isolates of E.canis, and suggested that in North America E.canis can be conservative (McBride et al., 1999, 2000).

Previous level studies have been flawed due to the lack of data on the cloning and characterization of new genes homologous immunoreactive proteins of Ehrlichia canis 28-kDa and a single multigene locus containing genes homologous protein of 28 kDa. In addition, previous research level is imperfect due to the lack of recombinant proteins such immunoreactive genes of Ehrlichia canis. This invention fills this long-standing need in the field.

SUMMARY of INVENTION

Some aspects according to the invention associated with the description of molecular cloning, sequencing, properties, and expression of genes homologous Mature immunoreactive proteins of Ehrlichia canis 28-kDa (marked P28-1, -2, -3, -5, -6, -7, -9) and identification of a single locus (10677-base pairs)containing nine genes proteins of Ehrlichia canis 28-kDa (P28-1 - P28-9). Eight P28 genes localized on one DNA strand and one genr found on the complementary chain. Homology of nucleic acids among the nine representatives of the P28 genes was 37-75%, and homology of amino acid sequences ranged from 28 to 72%.

One aspect according to the invention is associated with a DNA sequence encoding an immunoreactive protein of Ehrlichia canis in the 30 kDa. Preferably, the protein has an amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, 46, and gene has the sequence of nucleic acids selected from the group consisting of SEQ ID No. 1, 3, 5, 39, 41, 43, 45, and is representative of the polymorphic, numerous family genes. Typically, the protein has an N-terminal signal sequence that can be chipped off in the post-translational processing, which leads to the formation of a Mature protein of 28 kDa. In addition, genes encoding proteins of the 28-kDa, preferably are in the same multigene locus that is the same size 10677 base pairs and encodes nine homologous proteins of Ehrlichia canis 28-kDa.

In another aspect, the invention is associated with expressing a vector containing a gene that encodes an immunoreactive protein of Ehrlichia canis 28-kDa, and is able to Express the gene, when the vector is introduced into the cell.

In another aspect according to the invention presents a recombinant protein containing amino acid sequence selected from g is uppy, consisting of SEQ ID No. 2, 4, 6, 40, 42, 44 and 46. Preferably, if the amino acid sequence encoded by the sequence of nucleic acids selected from the group consisting of SEQ ID No. 1, 3, 5, 39, 41, 43, 45. Preferably, the recombinant protein contained four variable regions, which could be hydrophilic, antigenic and exposed on the surface. Recombinant protein can be used as the antigen.

In another aspect according to the invention presents a method of obtaining a recombinant protein, comprising the stage of receiving the vector, which contains expressing region containing a sequence that encodes amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44 and 46, are strongly related to the promoter; transfection of the vector into a cell; and culturing the cells under conditions effective for expression expressing the field.

The invention may also be described in some ways as a way of inhibiting infection with Ehrlichia canis from the subject, which includes identification of the subject prior to exposure to Ehrlichia canis or subject, suspect that he has been exposed to Ehrlichia canis or infected with Ehrlichia canis; and introducing the composition containing the antigen of Ehrlichia canis 28-kDa in the amount effective for inhibiting infection Ehrlichi canis. The inhibition can be accomplished by any means such as stimulation of humoral or cellular immune responses of the subject, or by other methods, such as the inhibition of the normal function of antigen 28-kDa or even compete with the antigen for interaction with any agent in the body of the subject.

Other and further aspects, features and advantages according to the invention will become apparent from the following description of preferred aspects of the invention, presented for the purpose of explanation.

BRIEF DESCRIPTION of DRAWINGS

To the main subject of discussion, which led the foregoing features, advantages and objectives of the invention, as well as other issues, would be established and understood in detail, a more detailed description of the invention, briefly summarized above, may be represented by references to certain aspects illustrated by the accompanying drawings. These drawings are part of the description. However, it should be noted that the accompanying drawings illustrate preferred aspects of the invention and are therefore not intended to be limiting thereof.

Figure 1 presents the sequence of the nucleic acid (SEQ ID No. 1) and deduced amino acid sequence (SEQ ID No. 2) gene P28-7, including the adjacent 5' and 3' nekojiru is the following sequence. The initiating ATG codon and the termination codon TAA marked in bold, and the 23-membered amino acid leader signal sequence is underlined.

Figure 2 shows the EF in PAG in the presence of DDS expressed recombinant fused with the P28-7-thioredoxin protein of 50 kDa (lane 1, arrow) and control thioredoxin 16-kDa (lane 2, arrow) and the corresponding immunoblot of recombinant fused with the P28-7-thioredoxin protein recognized by anti-E. canis serum dogs obtained in the recovery period (lane 3). Thioredoxin control is not detected by anticorodal E. canis (not shown).

Figure 3 presents a comparative analysis of the amino acid sequence of the protein P28-7 (ESA-1, SEQ ID No. 2), P28 protein-5 (C28SA2, partial sequence, SEQ ID No. 7), P28 protein-4 (C28SA1, SEQ ID No. 8), E.chaffeensis P28 (SEQ ID No.9), OMP-1 family E.chaffeensis (SEQ ID Nos: 10-14) and protein MAP-1 C.ruminantium (SEQ ID No. 15). Amino acid sequence of P28-7 is presented as a consensus sequence. Not represented amino acids identical P28-7 and are represented by points. Divergent amino acids are marked by the corresponding single-letter abbreviations. Gaps introduced to maximize alignment of amino acid sequences are shown as dashes. Variable regions are underlined and labeled VR1, VR2, VR3 and VR4). The arrows indicated the see on the predicted site of cleavage of the signal peptidase on the signal peptide.

Figure 4 schematically depicts the phylogenetic relationship P28-7 (USA-1), P28-5 (28SA2, partial sequence), P28-4 (28SA1) E.canis, representatives of a large gene family omp-1 E.chaffeensis, and protein map-1 C.rumanintium using the unbalanced structure of the tree. The length of each pair of branches represents the distance between the amino acid sequence of pairs. The line gives the opportunity to estimate the distance between sequences.

Figure 5 presents the data blot analysis Southern genomic DNA E.canis, completely split six individual restricteduse enzymes and hybridizing with DIG-labeled probe P28-7 (lanes 2-7); DIG-labeled taps molecular masses (tracks 1 and 8).

Figure 6 presents data comparing the predicted properties of proteins P28-7 (ESA-1, strain Jake) E.canis and P28 E.chaffeensis (Arkansas strain). Surface probability of predicted surface residues through the use of open Hexapeptide. Surface residue is any residue with >2.0 nm2water accessible surface area. Hexapeptide with a value greater than 1 was considered as the surface area. Antigenic index predicting antigenic determinants. Areas with a value above zero are possible antigenic determinants. T-cell motive determines the location of possible T-cell the antigenic determinants on the motif of the 5 amino acids 1 - glycine or polar residue, 2-hydrophobic residue, 3 - hydrophobic residue, 4 - hydrophobic or prolinnova residue and 5 - polar or glycine residue. The line indicates the position of amino acids.

Figure 7-1 7-2 presents the sequence of the nucleic acid and deduced amino acid sequences of protein coding genes E.canis 28-kDa: P28-5 (nucleotide 1-849: SEQ ID No. 3; amino acid sequence: SEQ ID No. 4) and P28-6 (nucleotide 1195-2031: SEQ ID No. 5; amino acid sequence: SEQ ID No. 6), including intergenic non-coding sequence (NC2, nucleotide 850-1194: SEQ ID No. 31). The initiating ATG codon and the termination codon is indicated in bold.

On Fig presents a schematic representation of the locus genes (5,592 Kb, containing five genes) proteins E.canis 28-kDa, demonstrating gene orientation and intergenic non-coding region (28NC1-4). Described genes protein of 28 kDa, as shown in locus 1 and 2 (shaded) (McBride et al., 1999; Reddy et al., 1998; Ohashi et al., 1998). Sequenced the complete sequence of P28-5, and the sequence of the new gene protein of 28 kDa labeled P28-6. Non-coding intergenic regions (28NC2-3) between P28-5, P28-6, and P28-7 completed the connection of previously unrelated loci 1 and 2.

Figure 9 schematically shows the phylogenetic relationship of the gene P28-4 (Ea28SA1), p28-5 (Ea28SA2), p28-6 (Ea28SA3), p28-7 (ESA-1) and p28-8 (USA-2) proteins E.canis 28-kDa based on the amino acid sequences using the unbalanced structure of the tree. The length of each pair of branches represents the distance between amino acid pairs. The ruler measures the distance between sequences.

Figure 10 presents a comparative analysis of intergenic non-coding nucleic acids sequences (SEQ ID Nos 30-33) protein gene E.canis 28-kDa. Not represented nucleic acids, indicated by dots (.), identical non-coding region 1 (28NC1). Divergence is indicated using the appropriate one-letter abbreviations. Introduced for maximal alignment of amino acid sequences of intervals is specified as a dash (-). Selected putative transcriptional promoter region (-10 and -35) and ribosomal binding site (RBS).

Figure 11 presents a schematic representation of the locus (10677-base pairs) of nine genes P28 E.canis, demonstrating genomic orientation and intergenic non-coding region. Genes P28 (P28-1, 2, 3, 9) (unshaded) identified in example 8. Shaded P28 genes were previously identified and marked as follows: P28-4. RA (Ohashi et al., 1998b) and ORF1 (Reddy et al., 1998); P28-5, P28-6 (McBride, et al., 2000); p28-7, p28 (McBride et al., 1999) and P30 (Ohashi et al., 1998b); and P28-8, P30-1 (Ohashi et al., 1998b).

On Figschematically shows the phylogenetic relationship P28-1 - P28-9 E.canis on the basis of amino acid sequences. The length of each pair of branches represents the distance is between amino acid pairs. The ruler measures the percentage of divergence between sequences.

On Figpresents the sequence of the nucleic acid (SEQ ID No. 39) and deduced amino acid sequence (SEQ ID No. 40) gene P28-1 E.canis.

On Figpresents the sequence of the nucleic acid (SEQ ID No. 41) and derived (base) amino acid sequence (SEQ ID No. 42) gene P28-2 E.canis.

On Figpresents the sequence of the nucleic acid (SEQ ID No. 43) and deduced amino acid sequence (SEQ ID No. 44) gene P28-3 E.canis.

On Figpresents the sequence of the nucleic acid (SEQ ID No. 45) and deduced amino acid sequence (SEQ ID No. 46) gene P28-9 E.canis.

DETAILED description of the INVENTION

The present invention describes the cloning, sequencing and expression of homologous genes encoding protein of 30 kilodaltons (kDa) Ehrlichia canis. Also a comparative molecular analysis of homologous genes of seven isolates of E. canis and multigene families omp-1 E.chaffeensis. Some new genes protein of 28 kDa identified as follows:

P28-7 (ESA-1) has an open reading frame of the 834-base pairs encoding a protein of 278 amino acids (SEQ ID No. 2) with a predicted molecular mass of 30.5-kDa. Identified N-terminal signal sequence, indicating that the protein under argueta posttranslational modification in the Mature protein 27.7-kDa.

P28-6 (28SA3) has an open reading frame of the 840-base pairs encoding a protein of 280 amino acids (SEQ ID No. 6).

Using PCR (PCR) for amplification of genes of proteins E.canis 28-kDa, completed the sequencing previously nezakonchennoy region P28-5 (28SA2). When analyzing the sequence of P28-5 detected open reading frame, 849 base pairs encoding a protein of 283 amino acids (SEQ ID No. 4).

PCR amplification using primers specific for intergenic non-coding regions of genes, proteins 28-kDa, led to the determination of the primary structure of the regions connecting the two previously separate locus, and thereby identified a single locus (5592-kb), which contains five genes (P28-4, -5, -6, -7 and -8) protein of 28 kDa. It was predicted that five proteins 28-kDa have signal peptides, whose removal leads to the formation of the Mature proteins, and have amino acid homology, ranging from 51%to 72%. Analysis of intergenic regions revealed a hypothetical promoter region for each gene, suggesting that these genes can be independently and differentially expressed. The size of intergenic non-coding regions (28NC1-4) ranged from 299 to 355 base pairs, and the homology is 48-71%.

In addition, there were sequenced previously unknown DNA in the reverse and forward direction of the above-mentioned locus, the C five consecutive genes P28, and identified P28-1, -2, -3 and-9. Therefore, the present invention identified the locus of the nine genes P28 E.canis, covering 10677 base pairs.

This invention, among other things, aims to study genes homologous protein P28 from Ehrlichia canis, P28-1, -2, -3, -6, -7 and P28-9, and the complete sequence of partially sequenced previously P28-5. Also opening is a multigene locus that encodes nine homologous proteins 28-kDa outer membrane of Ehrlichia canis. Eight of the P28 genes localized on a single DNA chain, and one of the P28 gene found on the complementary chain. Homology of nucleic acids among representatives of nine genes P28 ranges from 37 to 75%, and amino acid homology ranges from 28%to 72%.

According to this invention, the experts in this field can apply standard methods of molecular biology, Microbiology and techniques of recombinant DNA. Such methods are described in the literature in full. See, for example, Maniatis, Fritsch &Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis (M.J. Gait, ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames &S.J. Higgins, eds. (1985)]; "reduced and Translation" [B.D. Hames &S.J. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells and Enzymes [IRL Press, (1986)]; B. Perbal, "A Practical Guide to Molecural Cloning" (1984).

The invention provides for obtaining essentially pure DNA that encodes immunoreacted the hydrated protein of 28 kDa Ehrlichia canis. The protein encoded DNA according to the invention can have at least 80% sequence identity, preferably 85%, more preferably 90% and most preferably 95%) with the amino acids shown in SEQ ID No. 2, 4, 6, 40, 42, 44 or 46. More preferably, when the DNA contains the coding nucleotide sequence SEQ ID No. 1, 3, 5, 39, 41, 43, 45 or a degenerate variant of this sequence.

In this field it is well known that the amino acid sequence of the protein is determined by the nucleotide sequence of DNA which encodes a protein. Due to the degeneracy of the genetic code (i.e. most amino acids are encoded by more than one triplet (codon) of individual amino acids or polypeptide can be encoded different nucleotide sequences. Thus, the polynucleotide sequences of the invention under examination also contain the aforementioned degenerate sequences that encode the polypeptide according to the invention or a fragment or variant.

The invention also includes an essentially 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 area of nucleotide is, shown in SEQ ID No. 1, 3, 5, 39, 41, 43 or 45.

The term "essentially pure DNA" is meant DNA that is not part of the environment in which DNA is naturally found by separating the (partial or complete purification) from some or all of the molecules of such an environment or due to the restructuring of the sequences that flank the named DNA. Therefore, the term includes, for example, recombinant DNA, which is introduced into a vector that can replicate offline plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or DNA, which exists as a separate molecule (e.g., cDNA or a fragment of genomic or cDNA obtained by polymerase chain reaction (PCR; PCR) or cleaved restricteduse endonucleases)independent of other sequences. The term also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, for example a protein. Also included in this invention is a recombinant DNA which comprises a part of the nucleotides listed in SEQ ID No. 1, 3, 5, 39, 41, 43 or 45, which encodes an immunoreactive protein of Ehrlichia canis 28-kDa.

DNA must have at least 70% sequence identity with the coding sequence of the nucleotides presented in SEQID No. 1, 3, 5, 39, 41, 43 or 45, preferably at least 75% (e.g., at least 80%); and most preferably at least 90% identity. The identity between the two sequences is a direct function of the number of matching or identical provisions. If the position of the subunit in both of the two sequences is occupied by the same Monomeric subunit, e.g., if this position is occupied by adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in the sequence of 10 nucleotides in length are identical to the corresponding provisions in the second 10-nucleotide sequence, then the two sequences have 70% sequence identity. Typically, the length of comparison sequences is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably 75 nucleotides, and most preferably 100 nucleotides. Usually the identity of the sequences determined using software - analysis of sequence (for example, a software package - sequence analysis, Genetic computer group, Biotechnology center of the University of Wisconsin, 1710 University Avenue, Madison, WI 53705).

The present invention also considers the vector containing the coding DNA sequence, which contains a gene that encodes an immunoreactive protein of 28 kDa Ehrlichia canis, and named vector able to replicate in the host, which contains the operative coupling: a) origin of replication; b) a promoter; and C) a DNA sequence encoding a protein called. Preferably, the vector according to the invention contains a portion of the DNA sequence presented in SEQ ID No. 1, 3, 5, 39, 41, 43 or 45.

"Vector" can be defined as the replicated structure of nucleic acids, such as plasmid or viral nucleic acid. You can use vectors to amplify and/or Express nucleic acid encoding an immunoreactive protein of 28 kDa Ehrlichia canis. Expressing the vector is replicated design, in which the sequence of the nucleic acid encoding the polypeptide, is strongly related to a control sequence capable of effectively Express the polypeptide in the cell. The need for such control sequences varies depending on the chosen cell and the chosen method of transformation. Typically, the control sequences include a transcriptional promoter and/or enhancer, corresponding ribosomal mRNA binding sites, and sequences which control termination of transcripti and broadcast. Methods which are well known to specialists in this field can be used to construct expressing vectors containing appropriate transcriptional and translational control signals. See, for example, the methods described by Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (2ndEd.), Cold Spring Harbor Press, N.Y. Gene and its transcriptional control sequences identified as strongly related, if transcriptional control sequences effectively control the transcription of the gene. Not limited to, vectors according to the invention include plasmid vectors and viral vectors. Preferred viral vectors of the present invention are vectors derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes viruses.

Generally expressing vectors containing promoter sequences which facilitate the efficient transcription insertion of the DNA fragment, used in connection with the owner. Used in the description of the term "owner" is intended to encompass not only prokaryotes, but also eukaryotes, such as yeast, plants and animal cells. A recombinant DNA molecule or gene, which encodes an immunoreactive protein of Ehrlichia canis 28-kDa according to the invention, can be used to transformera the ü master, using any of the methods generally known to experts in this field. Particularly preferred is the use of a vector containing the coding sequence of the gene encoding an immunoreactive protein of 28 kDa Ehrlichia canis according to the invention for the purposes of prokaryotic transformation.

Prokaryotic hosts may include E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeast, such as Pichia pastoris, mammalian cells and insect cells. To achieve optimal cell growth transformed owners can ferment and cultivate ways that are known in this field.

Used in the description of the term "engineering"or "recombinant"cell refers to the cell in which you have entered a recombinant gene, such as the gene encoding the antigen of Ehrlichia canis. Therefore, engineering cells differ from naturally occurring cells that do not contain recombinante introduced gene. Thus, engineering the cells are cells that contain a gene or genes introduced by human hand. Recombinante introduced genes or will be in the form of a cDNA gene, the genomic copy of the gene, or will include genes, positional adjacent to the promoter by nature not associated with the particular introduced gene. In addition, the recombinant gene may be multi-the Rowan into the host genome, or may be placed in the vector or in the bacterial genome, transfetsirovannyh into the host cell.

The present invention also obtained essentially pure immunoreactive proteins 28-30 kDa E.canis, which contain amino acid sequences shown, for example, in SEQ ID No. 2, 4, 6, 40, 42, 44 or 46.

The expression "essentially pure protein" means a protein that has been separated from at least some of those components that accompany it in nature. Typically, a protein is considered essentially pure when it is at least 60% by mass, free from the proteins and other naturally occurring organic molecules with which it is naturally associated in vivo. Preferably the purity of the preparation is at least 75%, more preferably at least 90% and most preferably 99% by mass. Essentially pure immunoreactive protein of 28 kDa Ehrlichia canis can be obtained, for example, by extraction from a natural source; the expression of a recombinant nucleic acid that encodes an immunoreactive protein of 28 kDa Ehrlichia canis; or by chemical synthesis of the protein. Purity can be determined in any suitable way, for example by the method of column chromatography such as immunoaffinity chromatography using antibodies specific immunoreactive protein of 28 kDa Ehrlichia canis, polyacrylamide gel electrophoresis or analysis by the m HPLC. Protein is essentially free of naturally associated components when it is separated from at least some of those contaminants which accompany it in its original state. Thus, a protein that is chemically synthesized or obtained in a cellular system different from the cell from which he is by nature, will, by definition, essentially free from its naturally associated components. Accordingly, the essentially pure proteins include eukaryotic proteins synthesized in E. coli and other prokaryotes or any other organism in which they do not naturally occur.

In addition to a full-sized proteins, the invention also covers fragments (e.g., antigenic fragments) of immunoreactive protein of 28 kDa Ehrlichia canis (SEQ ID No. 2, 4, 6, 40, 42, 44 or 46). The term "fragment" refers to a polypeptide, which is usually at least 10 residues, more often at least 20 residues, and more preferably 30 (e.g., 50) residues in length, but less than full intact sequence. Fragments immunoreactive protein of 28 kDa Ehrlichia canis can be obtained by methods known to experts in this field, for example, by enzymatic digestion of naturally occurring or recombinant immunoreactive protein of 28 kDa Ehrlichia canis, using the methods of the s recombinant DNA, using expressing vector that encodes a defined fragment immunoreactive protein of 28 kDa Ehrlichia canis, or by chemical synthesis. The ability of the proposed fragment to show the properties of the immunoreactive protein of 28 kDa Ehrlichia canis (e.g., binding to an antibody specific against immunoreactive protein of 28 kDa Ehrlichia canis) can be evaluated methods are presented in the description.

Purified immunoreactive protein of 28 kDa Ehrlichia canis or antigenic fragment immunoreactive protein of 28 kDa Ehrlichia canis can be used for the formation of new antibodies or to test existing antibodies (e.g., as positive controls in the diagnostic study), using standard protocols known to specialists in this field.

As is well known in this field, this polypeptide may vary in its immunogenicity. Therefore, it is often necessary to bind the immunogen (e.g., a polypeptide according to the invention with a carrier. Typical and preferred carriers are hemocyanin clam (KLH) and serum albumin human. Means for connecting polypeptide with protein carrier are well known in this field and include glutaraldehyde, ester m-maleimidomethyl-N-hydroxysuccinimide, carbodiimide and bis-beastiary Benz is Dean. It is also clear that the peptide may be linked to protein genetic engineering methods, which are well known in this field.

Also in this area is well known that the immunogenicity against a specific immunogen can be enhanced through the use of non-specific stimulators of the immune response, known as adjuvants. Typical and preferred adjuvants include complete BCG, Detox (RIBI, Immunochem Research Inc.) ISCOMS and adjuvant is aluminium hydroxide (Superphos, Biosector).

In this invention included polyclonal antisera obtained, for example, in rabbits using immunoreactive protein of 28 kDa Ehrlichia canis or fragment immunoreactive protein of 28 kDa Ehrlichia canis as immunogen. Use standard protocols to obtain monoclonal and polyclonal antibodies, known to specialists in this field. Monoclonal antibodies obtained according to the aforementioned method, should be tested for their ability to identify recombinant clones cDNA Ehrlichia canis and to distinguish them from known cDNA clones.

The invention considers not only intact monoclonal antibodies, but also immunologically active fragment of an antibody such as a Fab fragment or (Fab)2; engineering the molecule is a single chain Fv; or a chimeric molecule such as an antibody, which contains a link to yuushuu specificity of one antibody, for example, the mouse of nature, and the remaining portions of another antibody, e.g., human.

In one aspect, the antibody or its fragment may be linked to a toxin or with a detectable label such as a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, an enzymatic label or a colorimetric label. Professionals in this field are well known labels listed and other suitable labels that can be used in accordance with this invention. Linking the mentioned labels with antibodies or their fragments can be carried out using standard methods, well known to specialists in this field.

Also assume that the pharmaceutical composition can be obtained by using new proteins according to the invention. In this case, the pharmaceutical composition includes a new active drug(s) according to the invention and a pharmaceutically acceptable carrier. The subject, which is the ordinary person skilled in the art can easily determine, without undue research, the appropriate dosages and routes of administration of the active ingredient according to the invention.

The phrase "pharmaceutically acceptable" refers to molecular particles or compositions that do not cause allergies and is and such an adverse reaction when administered to a subject. The preparation of aqueous compositions, containing a protein as an active ingredient, is fully explored in this area. Typically, such compositions receive in the form of injectable form, or in the form of liquid solutions or suspensions; also can be prepared solid forms suitable for solution or suspension in liquid prior to injection. The preparation also can be emulsified.

For the composition protein can be prepared in a neutral or salt form. Pharmaceutically acceptable salts include additive, acid salts (formed with free amino groups of the protein), and salts formed with inorganic acids, such as hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, almond, and the like. Salts formed free carboxyl groups can also be obtained with inorganic bases, such as hydroxides of sodium, potassium, ammonium, calcium or iron, and such organic bases as Isopropylamine, trimethylamine, histidine, procaine and the like.

When preparing the compositions of the solutions should be introduced in a way compatible with the dosage of the drug and in such a quantity that is therapeutically effective. Drugs are easily administered in a number of dosed four is, such as injectable solutions.

For example, for parenteral administration in an aqueous solution the solution should be suitably buffered if necessary and the liquid diluent first of all, made isotonic with sufficient amount of salt and glucose. Such special aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, in the light according to the invention a sterile aqueous medium, which can be applied, will be known to specialists in this field. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of liquid for subcutaneous injection either enter through infusion in the proposed area (see, for example, "Remington''s Pharmaceutical Sciences" 15ththEdition, pages 1035-1038 and 1570-1580). Some changes doses will inevitably be required depending on the status of a subject of which they treat.

In one aspect the present invention provides a DNA sequence encoding an immunoreactive protein of 30 kDa Ehrlichia canis. Preferably, the protein must have an amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, 46, and the gene must have a sequence of nucleic acids selected from the group consisting of SEQ ID No. 1, 3, 5, 39, 41, 43, 45, and to be representative of the polymorphic multig the frame family. More preferably, the protein has an N-terminal signal sequence that is removed in the post-translational processing, which leads to the formation of a Mature protein of 28 kDa. Even more preferably, DNA encoding the protein of 28 kDa, are located in the same multigene locus that is the same size 10677 base pairs and encodes nine homologous protein of 28 kDa Ehrlichia canis.

In another aspect of the present invention presents expressing a vector containing a gene that encodes an immunoreactive protein of 28 kDa Ehrlichia canis, and is able to Express the gene, when the vector is introduced into the cell.

In another aspect this invention provides a recombinant protein containing amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, 46. Preferably, the amino acid sequence encoded by the sequence of nucleic acids selected from the group consisting of SEQ ID No. 1, 3, 5, 39, 41, 43, 45. More preferably, the recombinant protein contains four variable regions, which are superficially located, hydrophilic and antigenic. Even more preferably, the recombinant protein is an antigen.

The following aspect of the present invention presents a method of obtaining a recombinant protein, comprising the stage of receiving, etc) is RA, which contains expressyou region comprising a sequence encoding a amino acid sequence selected from the group consisting of SEQ ID No. 2, 4, 6, 40, 42, 44, 46, functionally related to the promoter; the transfection of the vector into a cell; and culturing the cells under conditions effective for expression expressing the field.

Also in some aspects of the invention can be described a method of inhibiting infection with Ehrlichia canis in a subject, comprising the stages: identification of the subject, suspect that he has been exposed to or infected with Ehrlichia canis; and introducing the composition containing the antigen 28-kDa Ehrlichia canis in an amount effective to inhibit infection with Ehrlichia canis. The inhibition may be effected by any means, such as stimulation of humoral and cellular responses of the subjects, or in other ways, such as the inhibition of the normal function of antigen 28-kDa, or even compete with the antigen for interaction with some agent in the body of the subject.

The following examples are presented to illustrate various aspects of the invention and are not intended to limit the invention in any way.

EXAMPLE 1

Sequencing unknown 5'-

and 3'regions of the gene ECa28-1 (p28-7)

Erlichia and ochistnye Ehrlichia canis(strain Florida and Oleta Demon, DJ, Jake and Fuzzy) were provided by Dr. Edward Breitschwerdt (College of Veterinary Medicine, North Carolina State University, Raleigh, NC). E.canis (strain Louisiana) provided by Dr. Richard E.Corstvet (School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA) and E.canis (strain Oklahoma) provided by Dr. Jacqueline Dawson (Centers control and prevention, Atlanta, GA). Reproduction Herlihy carried out in DH82 cells with DMEM, supplemented with 10% bovine calf serum and 2mm L-glutamine at 37°C. Intracellular growth in DH82 cells was controlled by the presence of moral E.canis, using the methods of cytological staining. When 100% of cells were infected ehrlichiae, cells were collected, and then besieged by centrifugation at 17000 x g for 20 minutes. Cellular precipitation was twice destroyed in an ultrasonic homogenizer (Braun 2000-Sonic at 40V for 30 seconds on ice. Erlichia was purified as described previously (Weiss et al., 1975). The lysate was layered in discontinuous gradients renografin 42%-36%-30% and centrifuged at 80000 x g for 1 hour. Collected heavy and light zones containing erlichia, washed with sucrose-phosphate-glutamate buffer (SPG, 218 mm sucrose, 3.8 mm KN2RHO4, 7.2 mm To2NRA4, 4.9 mm glutamate, pH 7.0) and precipitated by centrifugation.

Obtaining nucleic acids.Genomic DNA of Ehrlichia canis received repeated suspendirovanie cleaned by renografin Herlihy in 600 μl of 10 mm Tris-HCl buffer (pH 7.5) with 1% dodecyl what Ulfat sodium (SDS, wt./about.) and 100 ng/ml of proteinase K as previously described (McBride et al., 1996). The resulting mixture was incubated for 1 hour at 56°and nucleic acids were extracted twice with a mixture of phenol/chloroform/isoamyl alcohol (24:24:1). DNA was besieged by absolute ethanol, washed once with 70% ethanol, dried and re-suspended in 10 mm Tris (pH 7.5). Plasmid DNA was purified using the kit for isolation of high purity plasmid (Boehringer Mannheim, Indianapolis, IN), and the PCR products were purified using the kit QIA-quick PCR purification (Qiagen, Santa Clarita, CA).

Cloning of the gene ECa28-1(p28-7) Full gene sequence P28-7 was determined using a universal set of Genome Walker Kit (CLONTECH, Palo Alto, CA), in accordance with the Protocol attached to the guide. Genomic DNA E.canis (isolate Jake) was completely digested with five restriction enzymes (DraI, EcoRV, PvuII, ScaI, StuI)to obtain DNA with blunt ends". Included in the set adapter (AP1) ligated to each end of the DNA E.canis. The genomic library was used as template to identify unknown DNA sequence of the gene P28-7 by PCR using a primer complementary to the known part of the sequence P28-7, and a primer specific to the adapter AP1. Specific to the P28-7 primers used for "walks" in the genome, were designed from the known sequence of the DNA obtained is ri amplification PCR P28-7 primers 793 (SEQ ID No. 16) and 1330 (SEQ ID No. 17). Primers 394 (5'-GCATTTCCACAGGATCATAGGTAA-3'; nucleotides 687-710, SEQ ID No.21) and S (5'-TTACCTATGATCCTGT GGAAATGC-; nucleotides 710-687, SEQ ID No. 22) used together with listed primer AP1 to amplify unknown 5' and 3' region of the gene P28-7 by PCR. The PCR product corresponding to the 5' region of the gene P28-7, amplified with primers C and AP1 (2000 base pairs), sequenced unidirectional primer C (5'-GAGTA ACCAACAGCTCCTGC-3', SEQ ID No. 23). The PCR product corresponding to the 3' region of the gene P28-7, amplificatory with primers 394 and AP1 (580 base pairs), sequenced bi-directional with the same primers. Non-coding region at the 5' and 3' regions that is adjacent to an open reading frame, sequenced and selected primers EC28OM-F (5'-TCTACTTTGCACTTCC ACTATTGT-3', SEQ ID No. 24) and EC28OM-R (5'-ATTCTTTTGCCACTATTT TTCTTT-3', SEQ ID No. 25), complementary to these regions in order to amplify the complete gene P28-7.

DNA sequencingDNA sequenced DNA sequencing machine (ABI Prism 377 (Perkin-Elmer Applied Biosystems, Foster City, CA). Whole genes P28-7 of the seven isolates of E.canis (four from North Carolina and one from Oklahoma, Florida, and Louisiana) amplified by PCR with primers EC28OM-F (SEQ ID No. 24) and EC28OM-R (SEQ ID No. 25) c temperature profile cycle 95°C for 5 min and 30 cycles of 95°C for 30 seconds, 62°C for 1 minute and 72°C for 2 minutes followed by continuation with 72°C for 10 minutes. According to the scientists bidirectional PCR products sequenced with the same primers.

EXAMPLE 2

PCR amplification, cloning, sequencing and expression of the gene ESA-1 (P28-7) E.canis

Expressing the vectorsA gene P28-7 amplified by PCR with primers ECOM-F and EC28OM-R and cloned into the cloning vector pCR2.1-TOPO TA to obtain the required set of sites of cleavage by restriction enzymes (Invitrogen, Carlsbad, CA). The insert was cut out from pCR2.1-TOPO with BstX 1 and ligated into the eukaryotic expressing vector rdnc 3.1 (Invitrogen, Carlsbad, CA), labeled RSDN/ES for further research. Plasmid RSDN/IS amplified and gene cut through the double splitting Cloned-XbaI and bidirectional were made in prokaryotic expressing vector pThioHis (Invitrogen, Carlsbad, CA). Clone (named pThioHis/EC) was produced recombinant protein fused with thioredoxin in Escherichia coli BL21. Recombinant protein was roughly purified in the insoluble phase by centrifugation. Control merged with thioredoxin protein was purified from the soluble cell lysates in native conditions, using spin-columns Nickel-NTA (Qiahen, Santa Clarita, CA).

Western blotting analysisRecombinant protein P28-7 E.canis were subjected to polyacrylamide gel electrophoresis in the presence of DDS (SDS) (SDS-PAGE) in a gradient gels (4-15% Tris-HCl (Bio-Rad, Hercules, CA) and transferred to pure nitrocellulose (Schleicher &Schuell, Keene, NH)using Polus the th cell transfer (Bio-Rad, Hercules, CA). The membrane was incubated with anticorodal obtained from infected E.canis dogs in the recovery period, diluted 1:5000 for 1 hour, washed, and then incubated with anti-dog IgG (H &L)conjugated with alkaline phosphatase affinity-purified secondary antibodies at a dilution of 1:1000 for 1 hour (Kirkegaard &Perry Laboratories, Gaithersburg, MD). Bound antibodies were visualized using the substrate 5-bromo-4-chloro-3-indolylacetic/tetrazole nitrosolobus (BCIP/NBT) (Kirkegaard &Perry Laboratories, Gaithersburg, MD).

The blotting analysis for SouthernTo determine whether the homology of numerous genes with the gene P28-7 in the genome E.canis, spent blot analysis for Southern, using a standard method (Sambrook et al., 1989). Genomic DNA E.canis was completely digested by each of the enzymes BanII, EcoRV, HaeII, and kpni restriction sites SpeI, which do not cut within the gene P28-7, and AseI, which were digested P28-7 at nucleotide 34, 43 and 656. The probe was received by PCR amplification with primers ECOM-F and ECOM-R and digoxigenin (DIG)-labeled deoxynucleotides (dNTPs) (Boehringer Mannheim, Indianopolis, IN) and were digested AseI. Split probe (566 base pairs) were separated by gel electrophoresis in agarose, purified on a gel and then used for hybridization. Fully digested genomic DNA E.canis were subjected to electrophoresis and transferred to nylon membrane (Boehringer Mannheim,Indianopolis, IN) and hybridized at 40°C for 16 hours with DIG-labeled probe gene P28-7 buffer DIG Easy Hyb, in accordance with the Protocol instructions (Boehringer Mannheim, Indianopolis, IN). Bound probe was determined using conjugated with alkaline phosphatase anti-DIG-antibody and the luminescent substrate (Boehringer Mannheim, Indianopolis, IN) and exhibited at the fine BioMax film (Eastman Kodak, Rochester, NY).

Analysis and comparison of sequencesDNAsequences of P28 E.chaffeensis and map-1 C.ruminantium received from the National Center for biotechnology information (NCBI). Analysis of the nucleotide and derived amino acid and protein sequences and phylogenetic analyses were performed with the software LASERGENE (DNASTAR, Inc., Madison, WI). Analysis of post-translational processing was carried out according to the method of McGeoch and von Heijne for recognition of signal sequences using the program PSORT (McGeoch, 1985; von Heijne, 1986).

The analysis of sequences of P28-7 from seven different strains of E.canis was performed with primers designed for amplification of the whole gene. The analysis showed that the sequence of this gene was found to be conservative in the number of isolates from North Carolina (four), Louisiana, Florida, and Oklahoma.

Results

Comparative analysis of sequences of nucleic acids of P28 E.chaffeensis and map-1 Cowdria ruminantium using the algorithm Jtun-Hein has established a coherent sequence with regions of high homology (> 90%). Detected homologous region (nucleotides 313-332 and 823-843 of map-1 C.ruminantium; 307-326 and 814-834 P28 E.chaffeensis) targetrole as areas annealing of primers for PCR amplification. PCR amplification of the gene P28-7 E.canis was carried out with primers 793 (5'-GCAGGAGCTGTTGGTTACTC-3') (SEQ ID No. 16) and 1330 (5'-CCTTCCTCCAAGTTCTATGCC-3') (SEQ ID No. 17), and receiving the resulting PCR product 518 base pairs. DNA E.canis amplified with primers 793 and 1330 with temperature profile cycle 95°C for 2 minutes and 30 cycles of 95°C for 30 seconds, 62°C for 1 minute, 72°C for 2 minutes followed by continuation 72°C for 10 minutes and extract with 4°C. the Sequence of nucleic acids of PCR product E.canis was obtained by sequencing of the product directly with primers 793 and 1330.

The sequence analysis revealed an open reading frame encoding a protein of 170 amino acids, and comparative sequence analysis of 518 base pairs, obtained by PCR amplification E.canis with the DNA sequence of the gene P28 E.chaffeensis revealed a similarity of more than 70%, indicating that the genes are homologous.

Adapter PCR with primers 394 and S conducted to detect the 5' and 3' segments of the sequence of the whole gene. Primer 394 formed four PCR product (3-TPN, 2-TPN, 1-TPN and 0.8-TPN) and the product of 0.8 base pairs sequenced bi-directional, using primers 39 and AP1. Deduced sequence overlapped with the 3' end of the product 518-base pairs, extending to 12-base pair open reading frame to the terminating codon. Also sequenced an additional 625 base pairs of noncoding sequence on the 3' end of the gene P28-7.

Primer IS used to amplify the 5' end of the gene P28-7, with the added primer AP1. Amplification with these primers resulted in three of PCR product (3,3-TPN, 3-TPN and 2-TPN). Fragment 2-TPN unidirectional sequenced with primer S. The sequence gave the prospective initiating codon of the gene P28-7 and completed open-reading frames 834 base pairs encoding a protein of 278 amino acids. Established additional 144 base pairs read sequence in the 5' non-coding region of the gene P28-7. Primers ECOM-F and EC28OM-R are chosen from the complementary non-coding regions, contiguous with the gene of the P28-7.

Amplificatory with these primers the PCR product sequenced directly using the same primers. The complete DNA sequence for the gene P28-7 E.canis (SEQ ID No. 1) is presented in figure 1. The PCR fragment P28-7, amplificatory with these primers contained the entire open-reading frames and 17 additional amino acids from the 5' non-coding region of the primer. Gene directed was subcloned into the expression is yuushi vector pThioHis, and E. coli (BL21) transformed this construct. Downregulation of P28-7-thioredoxin-fused protein was insoluble. Expressed protein had an additional 114 amino acids, associated with thioredoxin, 5 amino acids for enterokinase site recognition and 32 amino acids from the site multiple cloning 5' non-coding region primer for the N-end. Anticavity from an infected E.canis dogs received during the recovery period, recognize the expressed recombinant protein, but did not react with thioredoxin control (figure 2).

EXAMPLE 3

Homology sequences of the gene P28-7 E.canis

The sequence of the nucleic acid of the gene family P28-7 E.canis (834 base pairs) and omp-1 E.chaffeensis, including the signal sequence (P28-7, omp-1A, B, C, D, E and F), were aligned using the method Clustal (Clustal)to establish homology between these genes (comparative analysis not presented). Homology of nucleic acids remained equally (68.9%) and between P28-7 E.canis, P28 E.chaffeensis and omp-1F. Other putative genes of outer membrane proteins from the family of omp-1 E.chaffeensis, omp-1D (68,2%), omp-1E (66,7%), omp-1C (64,1%), map-1 Cowdria ruminantium (61,8%), gene 1 protein E.canis 28-kDa (60%) and gene 2 protein of 28 kDa (partial) (59,5%) also appeared to be homologous with the P28-7. OMP-1B E.chaffeensis were characterized by the lowest Homo what ohia nucleic acid (45,1%) P28-7 E.canis.

A comparative analysis of the predicted amino acid sequences of P28-7 E.canis. (SEQ ID No. 2) and P28 E.chaffeensis revealed amino acid substitutions, leading to the four variable regions (VR). Identified substitutions or deletions in the amino acid sequence and localization of the variable regions of the P28-7 E.canis and family MRA-1 E. chaffeensis (figure 3). Comparison of amino acid sequence, including the signal peptide, showed that the P28-7 E.canis shares the greatest homology with OMP-1F (68%) of the family of OMP-1 E.chaffeensis, followed by P28 E.chaffeensis (65,5%), MRA-1ST (65,1%), OMP-1D (62,9%), OMP-1C (62,9%), MAP-1 Cowdria ruminantium (59,4%), protein 1 28-kDa E.canis (55.6%) and protein 2 28-kDa (partial) (53,6%) and MRA-1B (43,2%). Analysis of phylogenetic relatedness on the basis of amino acid sequences showed that the P28-7 E.canis and MAP-1 C.ruminantium, proteins MRA-1 E.chaffeensis and proteins 1 and 2 (partial) 28-kDa E. canis are related (figure 4).

EXAMPLE 4

Predicted surface probability and immunoreactivity P28-7 E.canis

Analysis of P28-7 E.canis using profiles hydrobromide and hydrophilicity predicted surface-exposed region on the P28-7 (6). Eight major surface-exposed regions, consisting of 3-9 amino acids, were identified on the P28-7 E.canis and established the similarity with the profile surface-exposed regions on the P28 E.chaffeensis (6). Five more who Rupnik surface-exposed regions on the P28-7 E.canis localized in the N-terminal region of the protein. Surface-exposed hydrophilic region detected in all four variable regions P28-7 E.canis. Ten T-cell motifs were predicted in P28-7 when using the algorithm of Rothbard-Taylor (Rothbard-Taylor) (Rothbard and Taylor, 1988), and high antigenicity P28-7 E.canis predicted by the algorithm of antigenicity of the jameson-wolf (6) (Jameson and Wolf, 1988). Observed similarities in the antigenicity and T-cell motifs between P28-7 E.canis and P28 E.chaffeensis.

EXAMPLE 5

The definition of homologous genomic copies of the gene P28-7 E.canis

Genomic blotting analysis by Southern DNA E.canis, independently completely split restricteduse enzyme BanII, EcoRV, HaeII, Cloned, SpeI, which do not have endonuclease restriction sites in the gene P28-7, and AseI, which has an internal endonuclease restriction sites for nucleotides 34, 43 and 656, showed the presence of at least three homologous copies of a gene P28-7 (figure 5). Although P28-7 E.canis has internal parts of the AseI restriction, DIG-labeled probe used in the hybridization experiment, targetlevel region of a gene within a single DNA fragment formed by the cleavage of a gene by AseI. Splitting AseI gave 3 zones (approximately 566 base pairs, 850 base pairs and 3-TPN), which hybridized with the DNA probe P28-7, indicating the presence in the genome of numerous genes, homologous P28-7. When Ross the building by EcoRV and SpeI were formed two zones, which hybridized with the probe gene P28-7.

EXAMPLE 6

PCR Amplification of genes 28SA2 (p28-5), ECa28SA3 (p28-6) E.canis and identification of multigene locus

In order to specifically amplify the possible unknown genes forward ECa28SA2 (p28-5), were used for amplification primer range from 46f-specific P28-5 (5'-ATATACTTCCTACCTAATGTCTCA-3', SEQ ID No. 18), and primer 1330 (SEQ ID No. 17), which targetroot conserved region 3 conce gene P28-7. Amplificatory product was purified on gel and cloned into the cloning vector (Invitrogen, Santa Clarita, CA). Clone bidirectional sequenced with primers: M13 reverse the vector range from 46f, ECa28SA2 (5'-AGTGCAGAGTCTTCGGTTTC-3', SEQ ID No. 19), USA (5'-GTTACTTGCGGAGGACAT-3', SEQ ID No. 20). Amplified DNA with temperature profile cycle: 95°C for 2 minutes and 30 cycles of 95°C for 30 seconds, 48°C for 1 minute, 72°within 1 minute, followed by continuation 72°within 10 minutes, and the shutter speed 4°C.

The PCR product 2-TPN amplified with these primers, which contained 2 open reading frames. The first open reading frame consisted of a known region of a gene P28-5 and not previously sequenced the 3' part of the gene. In the forward direction from the P28-5 detected non-identical, but homologous gene of the protein of 28 kDa and marked ECa28SA3 (P28-6).

Specific primers indicated EaSA3-2 (5'-CTAGATTA GGTTATAGTATAAGTT-3", SEQ ID No. 26), the corresponding regions in the P28-6, and primer C (SEQ ID No. 23), which is attached to the field with the P28-7, was used to amplify the intergenic region between gene P28-6 and P28-7. Amplified DNA with temperature profile cycle: 95°C for 2 minutes and 30 cycles of 95°C for 30 seconds, 50°C for 1 minute, 72°within 1 minute, followed by continuation 72°within 10 minutes, and the shutter speed 4°C.

Amplified PCR product of 800 base pairs, which contained the 3' end of the P28-6, intergenic region between P28-6 and P28-7 (28NC3) and 5' end of the P28-7, connecting previously separate loci (Fig). The open reading frame 849 base pairs P28-5 encodes a protein of 283 amino acids, and P28-6 has an open reading frame 840 base pairs encoding a protein of 280 amino acids. Intergenic non-coding region between P28-6 and P28-7 was 345 base pairs in length (Fig.7 and 8).

EXAMPLE 7

Homology of nucleic acids and amino acids protein P28-4, P28-5, P28-6, P28-7, and P28-8 E.canis

Nucleic and amino acid sequences of all five genes of proteins E.canis 28-kDa were aligned using the method Clustal (Clustal), to investigate the homology between these genes. Homology of nucleic acids ranged from 58 to 75%, and similar amino acid homology, component from 67 to 72%, was observed among representatives of the genes is elkow E.canis 28-kDa (Fig.9).

Transcriptional promotor regionAnalyzed intergenic region between genes protein of 28 kDa relative to the promoter sequences by comparing agreed to the promoter regions of Escherichia coli and promoter of E.chaffeensis (Yu et al., 1997; McClure, 1985). Putative promoter sequences, including the field of RBS, -10 and -35 identified in 4 intergenic sequences corresponding to genes P28-5, P28-6, P28-7, and P28-8 (USA-2) (figure 10). Non-coding region in the opposite direction P28-4 (ECa28SA1) is unknown and has not been studied.

N-terminal signal sequenceAnalysis of amino acid sequences showed that a P28-7 E.canis has a deduced molecular mass of 30.5-kDa, and the deduced molecular mass of the whole P28-6 is 30.7-kDa. Both proteins have a predicted N-terminal signal peptide of 23 amino acids (MNCKKILITTALMSLMYYAPSIS, SEQ ID No. 27), which is similar to peptides predicted for the P28 E.chaffeensis (MNYKKILITSALISLISSLPGV SFS, SEQ ID No. 28) and MRA-1 protein family (Yu et al., 1999a; Ohashi et al., 1998b).

The preferred site of hydrolysis for the signal peptidase (SIS; Ser-X-Ser) (Oliver, 1985) installed at amino acid 21, 22 and 23 P28-7. Also discovered an additional the expected cleavage site in the amino acid position 25 (MNCKKILITTALISLMYSIPSISSFS, SEQ ID No. 29), is identical to the predicted site of cleavage P28 E.chaffeensis (SFS) and which positions the n to ensure the formation of Mature P28-7 with the predicted molecular mass of 27.7-kDa. The plot of the hydrolysis of the signal sequence of the previously described partial sequence of P28-5 predicted by the amino acid 30. However, analysis of signal sequences showed that the P28-4 has darassalam signal sequence.

Summary

Identified proteins with similar molecular mass and cloned from numerous ricketson agents, including E.canis, E.chaffeensis and C.ruminantium (Reddy et al., 1998; Jongejan et al., 1993; Ohashi et al., 1998). Previously described one locus in Ehrlichia chaffeensis c 6 homologous genes P28 and 2 locus in E.canis, each containing several homologous genes protein of 28 kDa.

The present invention has demonstrated the cloning, expression and properties of genes encoding Mature proteins E.canis 28-kDa, which are homologous multigene family of omp-1 E.chaffeensis and gene map-1 C.ruminantium. Identified two new gene protein of 28 kDa, P28-7, and P28-6. The present invention fully sequenced another gene proteins E.canis 28-kDa, P28-5, partially sequenced previously (Reddy et al., 1998). Also carried out the identification and characterization of a single locus in E.canis, containing five genes proteins E.canis 28-kDa (P28-4, P28-5, P28-6, P28-7, and P28-8).

Proteins E.canis 28-kDa are homologous to a family of MRA-1 E.chaffeensis and squirrel MAR 1 C.ruminantium. The most homologous proteins E.canis 28-kDa (P28-6, P28-7, and P28-8), location is us in the locus. Homology named proteins ranged from 67.5% to 72.3%. The divergence among these proteins in the 28-kDa ranged from 27.3% to 38.6%of. Proteins E.canis 28-kDa, P28-4, P28-5 was the least homologous with homology component from 50.9% to 59.4 per cent, and the divergence from 53.3 percent to 69.9 per cent. Differences between genes belong mainly to the four hypervariable regions and suggest that these areas are exposed on the surface and subjected to selective exposure of the immune system. Described conservatism P28-7 among the seven isolates of E.canis (McBride et al., 1999), suggesting that E.canis may be clonal in North America. On the contrary, described a significant variety P28 among isolates E.chaffeensis (Yu et al., 1999a).

It turned out that all of the proteins E.canis 28-kDa as a result of posttranslational modifications turn from protein 30-kDa Mature protein of 28 kDa. Was recently identified signaling sequence P28 E.chaffeensis (Yu et al., 1999a), and sequencing of N-terminal amino acid sequence confirmed that the protein undergoes post-translational modification, which leads to cleavage of the signal sequence with the formation of the Mature protein (Ohashi et al., 1998). The assumption is that the leader sequence of the OMP-1F and OMP-1E also have the leader signal peptide (Ohashi et al., 1998). The signal sequence is certifitsirovannyie on OMP-1F and OMP-1E E.chaffeensis and P28, are homologous leader sequences of protein E.canis 28-kDa. The promoter sequence for the P28 genes were not detected experimentally, but presumed promoter region identified by comparing with the coordinated sequences of the promoter regions of RBS, -10 and -35 E.coli and other Herlihy (Yu et al., 1997; McClure, 1985). Such promoter sequences allow each gene potentially be transcribed and translated, indicating that these genes can be differentially expressed in the host. Persistence of infection in dogs may be associated with differential expression of genes P28, leading to antigenic changes in vivo, thus providing the microorganism the ability to evade the immune response.

Discovered that the genes of proteins E.canis 28-kDa take the homology of the nucleic acid sequences and amino acid sequences with family genes omp-1 E.chaffeensis and genome map-1 C.ruminantium. Previous research has identified a protein of 30 kDa E.canis, which reacts with anticorodal against E.chaffeensis obtained during the recovery period, but it is believed to differ in antigenic properties (Rikihisa et al., 1994). Conclusions based on comparisons of amino acid substitutions in the four variable regions of proteins E.canis 28-kDa, confirmed e is the opportunity. At the same time, the findings also suggest that the amino acids responsible for antigenic differences between P28 E.canis and E.chaffeensis, localized in the above-mentioned variable areas and easily accessible to the immune system.

Described that the immunoreactive peptides localized in the variable regions of proteins C.ruminantium, E.chaffeensis E.canis and 28-kDa (Reddy et al., 1998). Analysis of P28 E.canis and E.chaffeensis showed that all of the variable regions are predicted by superficial amino acids. A study in dogs showed no cross immunity between E.canis and E.chaffeensis (Dawson and Ewing, 1992). The results of the observations can be associated with antigenic differences in variable areas P28, and other immunologically important antigens of these species Herlihy. In another study it was shown that anticavity person obtained from infected E.chaffeensis patients during the recovery period, recognized a protein(and) 29/28-kDa E.chaffeensis and interacted with homologous proteins E.canis (Chen et al., 1997). It turned out that homologous and gives cross-react epitopes on the protein of 28 kDa E.canis and P28 E.chaffeensis recognized by the immune system.

Proteins E.canis 28-kDa can be important immunoprotective antigens. Several reports have demonstrated that the antigen E.canis 30-kDa shows yet strong immunoreactivity (Rikihisa et al., 1994; Rikihisa et al., 1992). Antibodies to anticigarette obtained from humans and dogs during the recovery period, reacted with a protein of this size from E.chaffeensis and E.canis, suggesting that they may be important immunoprotective antigens (Rikihisa et al., 1994; Chen et al., 1994; Chen et al., 1997). In addition, antibodies to proteins of 30, 24 and 21 kDa was developed early in immuno response to E.canis (Rikihisa et al., 1994; Rikihisa et al., 1992), indicating that these proteins may be especially important in immune responses in the acute form of the disease. Recently, a family of homologous genes encoding proteins of the outer membrane with a molecular mass of 28 kDa, were identified in E.chaffeensis, and found that in mice immunized with recombinant P28 E.chaffeensis, was developed immunity against infection homologues (Ohashi et al., 1998). It is shown that the P28 E.chaffeensis is present in the outer membrane, and immunoelectron microscopy localized P28 on the surface of the microorganism, and thus suggesting that it may function as adhesin (Ohashi et al., 1998). It is possible that proteins E.canis 28-kDa identified in this study have the same localization and may perform a similar function.

Comparison of the P28-7 from different strains of E. canis showed that the gene is obviously completely conservative. The studies performed on E.chaffeensis, trademonster is believed immunological and molecular evidence of diversity. Patients infected E.chaffeensis, are characterized by variable immunoreactivity against proteins 29/28-kDa, indicating antigenic diversity (Chen et al., 1997). Recently at the molecular level data were obtained, which are proof of antigenic diversity in the P28 gene of E. chaffeensis (Yu et al., 1999a). Comparison of the five isolates of E. chaffeensis has revealed that two isolates (Sapulpa and St. Vincent) had 100% identity, but the other three (Arkansas, Jax, 91HE17) differed to 13.4% at the amino acid level. Conservatism P28-7 E.canis suggests that strains of E.canis found in the United States, may be genetically identical and, thus, protein E.canis 28-kDa is an attractive candidate for vaccine regarding erlichiosis dogs in the United States. Further analysis of the isolates E.canis outside the United States can provide information regarding the origin and evolution of E.canis. Conservatism protein of 28 kDa makes it an important potential candidate for reliable serodiagnosis of erlichiosis dogs.

Currently, the role of multiple homologous genes is not known; however, the resilience of infections E.canis in dogs can be expected to be associated with antigenic variability due to variable expression of the homologous gene of the protein of 28 kDa, thus allowing E.canis to evade and monogo control. The variability of genes msp-3 in A. marginale is partly responsible for the variability in the protein MSP-3, resulting in sustainable infections (Alleman et al., 1997). Studies of gene expression protein of 28 kDa by E.canis in acutely and chronically infected dogs will provide understanding of the role of gene family protein of 28 kDa in the persistence of infection.

EXAMPLE 8

Identification of genes P28-1, P28-2, P28-3, P28-9 E.canis

Sequenced unknown DNA in the reverse and forward direction of the locus, of the five consecutive P28 genes described above, using the selected specific gene primers for P28-1 (ESA-75S) and P28-5 (ESA-5-818f to undergo a genetic locus in two directions. To install an unknown sequence, multiple screenings gene locus was carried out as follows: 1,9 TPN in the forward direction 5-gene locus amplified and sequenced using primers P28-5-818f-(5'TTA AAC ATA TGC CAC TTC GCA CTA-3', SEQ ID No. 34), receiving amplicon 900 base pairs, and 1191 (5'-TAT GAT CGT GTA AAA TTG CTG TGA GTA T-3', SEQ ID No. 35), receiving amplicon 1-TPN 3,67 TPN DNA in the forward direction of the locus of the five genes and amplified, sequenced with primers ESA-75S (5'TAC TGG CAC GTG CTG GAC TA-3', SEQ ID No. 36), receiving amplicon 1,6-TPN; ESA'-1600 (5'-CAC CAA TAA ATG GAG AGA CTT C-3', SEQ ID No. 37) c the formation of the amplicon 1,6-TPN; and 3125 (5'-AAT CCA TCA TTT CTC ATT ACA GTG TG-3', SEQ ID No. 38), and getting plicon 800 base pairs. The locus of the nine tandemly arranged genes, consisting of four new genes P28 and five P28 genes described above, meant P28-1 to P28-9 (11).

Were compared to the sequences of the nucleic acid and amino acid sequences of P28 E.canis, using the method Clustal (Clustal), to investigate the homology between these genes. Homology named proteins ranged from 67.5% to 75%, and the divergence between these proteins P28 was 26.9%-38%. Protein P28 E.canis, P28-1, P28-2, P28-9 at least appeared to be homologous with other P28 genes with homology from 37% to 49% and divergence 53-77%. Homology of nucleic acids nine P28 genes ranged from 28 to 72%. The phylogenetic relationship P28 E.canis, on the basis of the amino acid sequence presented on Fig.

The nucleotide sequence and the number of deposits.Number of Deposit GenBank for sequences of nucleic acid and amino acid sequences of the locus of the nine genes P28 E.canis (strain Jake) is AF082744. Initially, the number of the Deposit was intended for P28-7, but was upgraded to the sequence of the locus of the nine genes P28, which includes P28-7. Rooms deposits GenBank for sequences of nucleic acid and amino acid sequences of P28-7 in other isolates E.canis described in this study are the I: Louisiana, AF082745; Oklahoma, AF082746; Demon, AF082747; DJ, AF082748; Fuzzy, AF082749; Florida, AF082750.

Many areas in the field of 28 kilodaltons were identified through immunoblots in the sera of infected E.canis dogs taken during the recovery period (Rikihisa et al., 1994), and the expression of many proteins P28 may be the explanation of the obtained data. The blot analyses for Southern indicate that other genes P28, in addition to the five representatives of the considered locus, present in the genome (McBride et al., 1999; Ohashi et al., 1998b).

In this study identified a single gene locus, containing nine consecutive genes P28 E.canis encoding homologous but non-identical genes P28. The locus of the nine genes includes four new P28 gene (Fig-16) and five tandemly arranged genes P28, which are described above. Eight of the P28 genes localized on a single DNA chain, and one of the P28 gene found on the complementary chain. Homology of nucleic acids among the nine representatives of the P28 genes was 37-75%, and amino acid homology ranged from 28 to 72%.

Found that the P28 E.canis are closely related protein of 28 kilodaltons other species such as E.chaffeensis to which they relate (McBride et al., 2000). Differences among the proteins found mainly in some of the major hypervariable regions and indicate that these areas are the stand is Resto exposed and subjected to selective exposure of the immune system (McBride et al., 2000).

Among seven geographically different isolates described conservatism P28 gene (P28-7) E.canis (McBride et al., 1999), suggesting that E.canis may be highly conserved in North America. Similarly, glycoprotein E.canis 120-kDa was also conservative among isolates in the United States (Yu et al., 1997). On the contrary, genes as a 120-kDa and the protein of 28 kDa E.chaffeensis were divergent among isolates (Yu et al., 1999a; Chen et al., 1997). It turned out, the diversity of genes proteins E.chaffeensis 28-kDa is the result of point mutations in the hypervariable regions, possibly due to selective immune effects (Yu et al., 1999a). The data presented suggest that E.canis was introduced to North America relatively recently, and this may be the explanation of conservatism, which was observed among the isolates. Conservatism P28 genes in isolates of E.canis can provide an opportunity for the development of vaccines and serodiagnostics antigens, which are especially effective for disease prevention and serodiagnosis. The mixture P28 can provide the most reliable serodiagnostic test, however, described that serological diagnosis with one P28 can be used for immunoassay (Ohashi et al., 1998b; McBride et al., 1999).

The following references are cited in the description.

Any patents or publications mentioned in this specification indicate the professional level of specialists in this area, which are relevant to the invention. The above-mentioned patents and publications are entered into the description by reference to the same extent as if each individual publication were individually presented to the incorporation by reference.

The person skilled in the art will readily appreciate that the invention is conveniently adapted to carry out the objectives and obtain the above mentioned results and benefits, and perform their own tasks. These examples along with the methods, procedures, treatments, molecules, and specific compounds represented in the description, in the present time are typical preferred aspects, are exemplary and are not intended to restrict the framework of the invention. Change aspects and other applications that require specialists in this field will correspond to the essence of the invention defined by the scope of the claims.

1. Isolated DNA sequence encoding a protein of Ehrlichia canis in the 30 kilodaltons, where this protein is immunoreactive against serum against Ehrlichia canis and where this protein contains an amino acid sequence selected from the group consisting of SEQ ID No.40, 42,44 and 46.

2. The DNA sequence according to claim 1, where this protein has an N-terminal signal sequence.

3. The DNA sequence according to claim 2, where the specified protein excision of modified protein in the 28 kilodaltons.

4. The DNA sequence according to claim 1, where the specified DNA contains a sequence selected from the group consisting of SEQ ID No.39, 41, 43 and 45.

5. The DNA sequence according to claim 1, where the specified DNA contained in a single locus Ehrlichia canis.

6. The DNA sequence according to claim 5, where the specified locus represents a multigene locus of 10677 base pairs in length.

7. The DNA sequence according to claim 6, where the specified locus contains genes encoding homologous proteins of Ehrlichia canis 28-kilodaltons.

8. The DNA sequence according to claim 7, where these homologous proteins of Ehrlichia canis 28-kilodaltons selected from the group consisting of P28-1, P28-2, P28-3, P28-4, P28-5, P28-6, P28-7, P28-8, P28-9.

9. A vector containing a DNA sequence according to claim 1, which encodes the protein of 30 kilodaltons, immunoreactive against serum against Ehrlichia canis.

10. The vector according to claim 9, where the specified vector is expressing vector that can Express the peptide or polypeptide encoded by a sequence selected from the group consisting of SEQ ID No.39, 41, 43 and 45 when the specified expressing vector introduced into the cell.

11. Recombinant protein Ehrlicha canis 28 kilodaltons, containing the amino acid sequence selected from the group consisting of SEQ ID No.40, 42, 44 and 46, and this protein is immunoreactive against serum against Ehrlichia canis.

12. Recombinant protein according to claim 11, where the specified amino acid sequence encoded by a nucleic acid segment containing a sequence selected from the group consisting of SEQ ID No.39, 41, 43 and 45.

13. A host cell containing a nucleic acid segment selected from the group consisting of SEQ ID No.39, 41, 43 and 45.

14. A method of obtaining a recombinant protein according to claim 11, which includes stage

obtaining a vector that contains expressing region comprising the sequence encoding amino acid sequence selected from the group consisting of SEQ ID No.40, 42, 44 and 46, operatively associated with a promoter; transfection of the indicated vector into a cell; and culturing the specified cells under conditions effective for expression of the indicated expressing the field.

15. The antibody, immunoreactive against the polypeptide containing the amino acid sequence selected from the group consisting of SEQ ID No.40, 42, 44 and 46.

16. Method of inhibiting infection with Ehrlichia canis from the subject, which includes identification of the subject prior to exposure to Ehrlichia canis or agent suspected in t is m, he has been exposed to Ehrlichia canis, or infected with Ehrlichia canis; introducing a composition containing the antigen of Ehrlichia canis 28-kDa in the amount effective for inhibiting infection with Ehrlichia canis.

17. The method according to clause 16, where the specified antigen 28-kDa is a recombinant protein containing amino acid sequence selected from the group consisting of SEQ ID No.40, 42, 44 and 46.

18. The method according to 17, where the indicated recombinant protein is encoded by gene containing a sequence selected from the group consisting of SEQ ID No.39, 41, 43 and 45.

19. The method according to clause 16, where the specified composition comprising an antigen in the 28-kDa, is distributed in a pharmaceutically acceptable carrier.



 

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13 cl, 3 dwg, 1 tbl

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