Immunodominant antigenic protein mass of 120 kda and the gene of ehrlichia canis

 

The invention relates to biotechnology and represents the gene of the protein mass of 120 kDa Ehrlichia canis, amplificatory by PCR using primers derived from the DNA sequences flanking the gene of the protein mass of 120 kDa Ehrlichia chaffeensis. Recombinant protein mass of 120 kDa E. canis contains 14 tandem repeated units, each of which consists of 36 amino acids. These repeating units are hydrophilic and is assumed to be surface-exposed. Recombinant protein mass of 120 kDa E. Canis is also antigenic and reacts with serum produced in dogs recovering from dog erlichiosis. This invention allows to inhibit the infection, caused by Ehrlichia canis, by introducing a composition containing the antigen of E. canis, 5 S. and 7 C.p. f-crystals, 11 tab., 1 PL.

The scope of the invention

In General terms, the present invention relates to the field of molecular biology of bacteria, parasites, and in particular to agents that cause disease type ricketsiosis, and bacteria Ehrlichia. More specifically the present invention relates to the molecular cloning and characterization of the gene immunoreactive protein with a mass of 120 kDa Ehrli the global bacteria, living in endosome hematopoietic cells and infect a variety of animal hosts, including humans, domestic and wild dogs, deer, horses, sheep, cattle, and wild rodents. Each clan member Ehrlichieae has its own tropism towards target cells. Most species of Ehrlichia are either monocytotropic (E. canis, E. chaffeensis, E. sennetsu, E. risticii and E. muris), or granulocytopenia (human granulocytic Ehrlichia, E. eqvi, E. phagocytophila and E. ewingii) except Cowdria ruminatium, which develops in the endothelial cells of the host, and Anaplasma marginale, which erythrocytic parasite.

Although the bacteria Ehrlichia were already described in the beginning of this century, but in the United States until the last decade, they had attracted the attention of researchers because they are considered pathogens of importance only for veterinary use. A newfound interest in erlichia due to the occurrence of erlichiosis slaying a man. In the last decade in the United States were opened two new pathogen Ehrlichia (E. chaffeensis and E. phagocytophila-like microorganism person) (Bakken J. S. 1994, Chen S. M., 1994, J. C. M., Fishbein D. C., 1987, Maeda, K., 1987). Ehrlichia canis, the prototype species of this genus, is an etiological factor rd arlais is a disease widespread throughout the world and portable dog tick Rhipicephalus sanguineus (Groves M. G., 1975, Lewis G. E. Jr., 1977). Ehrlichia canis is a sharp moderate and transient increase in temperature, which may progress with the development of severe disease and syndrome with fatal outcome (tropical pancytopenia dogs) (Buhles, W. C., 1974, Greene, C. E. & J. W. Harvey, 1984, Walter, J. S. 1970). Worldwide cost of treatment of Pets and service dogs infected with E. canis, make millions of dollars per year. In addition, the bacterium E. canis also endangers people's health. Ehrlichia canis or antigenic unidentifiable microorganism was recently isolated from humans (Perez M, 1996).

Identifying the genetic and antigenic composition of E. canis is the main factor for studying the pathogenesis of erlichiosis dogs and the development of effective vaccines. Its genetic and antigenic properties of the bacterium E. canis is closely related bacteria E. chaffeensis (Anderson C. E., 1991, 1992, Chen S. M. f Am. J. Trop. Med. Hyg). So dog arlais may be a suitable model for studying the pathogenesis monocytotropic Ehrlichia species, including E. chaffeensis.

Previous works have the disadvantage that they do not reported on the cloning and characterization of immunoreactive immunoreactive protein with a mass of 120 kDa Ehrlichia canis. The present invention fulfills this long-standing need and requirements in this field of technology.

Brief description of the invention

In one variation of its implementation of the present invention relates to gene, codereuse immunoreactive protein mass of 120 kDa Ehrlichia canis. This protein mass of 120 kDa preferably has the amino acid sequence of SEQ ID NO:8, and its gene has the nucleotide sequence SEQ ID NO:7.

In a preferred variant of its implementation of the present invention relates to expressing the vector containing the gene encoding immunoreactive protein mass of 120 kDa Ehrlichia canis, where specified expressing vector when introduced into the cell is able to Express this gene.

In another embodiment, the present invention relates to a recombinant protein containing the amino acid sequence of SEQ ID NO:8. Preferred is an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7. The indicated recombinant protein mass of 120 kDa preferably contains 14 tandem repeated units, each of which contains 36 amino acids. More preferably, if the repeat is Oh 120 kDa antigen is.

In a preferred embodiment, the present invention relates to a method for producing a recombinant protein with a mass of 120 kDa, including the stage of receiving the vector containing expressing region having a sequence encoding the amino acid sequence of SEQ ID NO:8, is correctly connected to the promoter; transfection of the indicated vector into a cell; and culturing the cell under conditions effective for the expression of expressing this area.

The present invention may be described in some embodiments of implementation relating to a method of inhibiting infection caused by Ehrlichia canis in the subject, including the identification stage of the subject, presumably exposed to bacteria or infected by the bacterium Ehrlichia canis; and introducing the composition containing the antigen mass of 120 kDa Ehrlichia canis in the amount effective for inhibiting infection caused by Ehrlichia canis. Such inhibition may be achieved by any means, such as, for example, stimulation of this subject humoral or cellular immune response, or any other means, such as inhibition of the normal function of antigen mass of 120 kDa, or Yes and additional aspects, distinguishing features and advantages of the present invention will be apparent from the following description of preferred embodiments of the present invention, shown for purposes of description.

Brief description of drawings

To the above distinctive features, advantages and objectives of the present invention, etc. have been more clear in detail, the description of the present invention, summarized above, may be more specifically illustrated by some variants of its implementation with reference to the accompanying drawings. These drawings are part of the present description. However, it should be noted that these drawings illustrate the preferred variants of the present invention, and therefore should not be construed as limiting its scope.

In Fig.1 shows the DNA sequence and position of oligonucleotide primers derived from the gene of the protein mass of 120 kDa E. chaffeensis (where there's no shading frame), and DNA sequences flanking the gene (line). Positions of primers are indicated as advantages and disadvantages for DNA sequences that are located above and below the gene of the protein mass of 120 kDa, respectively. It was constructed nine the primers were used for amplification of the gene of the protein mass of 120 kDa E. canis by PCR.

In Fig.2A shows the agarose gel electrophoresis carried out for the repeating units of the gene of the protein mass of 120 kDa E. canis. Plasmid RSA was first hydrolysed by the enzyme EcoRI to obtain a paste, and then she was hydrolysed by the enzyme SheI through various time intervals. In Fig.2B shows the definition of the number of repetitions by the method of southern blotting. DNA gidralizovanny within 35 minutes by enzyme Shel, was transferred from the gel in panel And on nylon membrane and hybridized with an oligonucleotide probe labeled with digoxigenin, which was hybridisable with DNA sequences upstream of the repeats of the gene of the protein mass of 120 kDa E. canis. ND = non-hydrolyzed DNA.

In Fig.3 shows the DNA sequence of the gene of the protein mass of 120 kDa E. canis (SEQ ID NO: 7) and deduced amino acid (SEQ ID NO:8). Nucleic acid repeats 1, 3, 5, 7, 9, 11 and 13 are underlined.

In Fig.4 shows a phylogenetic tree of the repeating units of the gene of the protein mass of 120 kDa E. canis. The scale represents the % difference in the DNA sequence.

In Fig.5 illustrates a comparative analysis of the primary amino acid sequence of proteins by mass of 120 kDa E. canis (SEQ ID NO:10) and E. chaffeensis (SEQ ID NO:9). The dashes indicate the IDA is financing on the surface hydrophobicity of proteins by mass of 120 kDa E. canis, E. chaffeensis. The wedge-shaped area between the arrows is the domain of repetitions. All repetitions in both proteins are hydrophilic and surface exposed. The second recurring unit of the protein mass of 120 kDa E. canis and the peak of the first repeating unit of the protein mass of 120 kDa E. chaffeensis are located between the two arrows and more coarsely shown in Fig.7.

In Fig.7 shows a comparison of surface-exposed amino acids in the repeating unit of the protein mass of 120 kDa E. canis and E. chaffeensis. In Fig.7A shows the probability of exposure of amino acids on the surface. The corresponding region specified by two wedge-shaped arrows in Fig.6. Letters in bold, conservative amino acids for E. canis and E. chaffeensis. In Fig.7B shows a comparison of the primary amino acid sequence represented in panel A (SEQ ID NO:11-12). Dashes indicate identical amino acids. Dots indicate conservative substitutions.

In Fig.8 shows the agarose gel electrophoresis, performed for the gene of the protein mass of 120 kDa strain of E. canis, hydrolyzed SI. Recombinant plasmid pCR2.1 were first hydrolyzed with EcoRI for selecting insert from the vector, and then partially hydrolyzed with SpeI. Digitalizovane: DNA gene belkasmi insert.

In Fig.9A shows SDS page-CIS-electrophoresis of the protein mass of 120 kDa E. canis, expressed in E. coli. 1, GST-hybrid protein mass of 120 kDa; 2, recombinantly protein mass of 120 kDa E. canis, derived from GST-hybrid protein thrombin. In Fig.9B shows the agarose gel electrophoresis for the plasmid pGEX expressing a protein mass of 120 kDa E. canis. The insert arrow.

In Fig.10 shows Western blot analysis of mouse antisera against recombinant protein with a mass of 120 kDa E. canis-reactive antigen of E. canis (lane 1) and recombinant protein with a mass of 120 kDa E. canis (lane 2, arrow).

In Fig.11 shows Western blot analysis of serum recovering dogs reacting with recombinant protein with a mass of 120 kDa E. canis.

Detailed description of the invention

In accordance with the present invention can be used standard methods of molecular biology, Microbiology and recombinant DNA technology known to specialists. These methods are described in detail in the literature. For Example, Maniatis, Fritish & 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 have values, defined below.

These amino acids are preferably "L"isomeric form. However, L-amino acid residues can be replaced by the "D"isomeric forms, provided that the desired functional properties of the polypeptide in respect of binding to immunoglobulin will remain unchanged. NH2means the free amino group present at the amino end of the polypeptide. COOH means free carboxypropyl present carboxylic polypeptide. In accordance with the standard nomenclature of polypeptid, J. Biol.Chem. 243:3552-59 (1969), indicate amino acid residues represented in the mapping table.

It should be noted that all sequences of amino acid residues represented by formula, the left and right orientation which is normal to the direction from the amino end to the carboxy-end. In addition, it should be noted that a dash at the beginning or at the end of the sequence of amino acid residues indicates a peptide bond with another sequence of one or more amino acid residues. The table presents for identifying the three-letter and one-letter codes that meet this description.

Term is t as an Autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. The term "vector" means a replicon, such as plasmid, phage or cosmid, to which may be attached to another segment of DNA, was carried out to replicate this attached segment.

The term "DNA molecule" means a polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) or in their single-stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. When discussing the structure in accordance with the standard legend indicates only the sequence in the direction of 5’3’ along retranscribing DNA chain (i.e., the chain having a sequence homologous to the mRNA sequence).

The term "site of replication initiation" means DNA sequences that are involved in DNA synthesis. "Coding sequence" DNA is duhamic the stay, transcribed and translated in vivo with the formation of the polypeptide. The boundaries of the coding sequence are determined by the start-codon at the 5’ amino-end and codon translation termination at the 3’ carboxy-end. The coding sequence can be, but are not limited to, sequences of prokaryotes, cDNA from mRNA of eukaryotes, genomic DNA sequences from the DNA of eukaryotes (e.g., a mammal), and even synthetic DNA sequences. The polyadenylation signal and the sequence termination of transcription are typically located 3’-end with respect to the coding sequence.

Transcriptional and translational regulatory sequences are regulatory DNA sequences, such as promoters, enhancers, polyadenylation signals, terminators, etc., that provide for expression of the coding sequence in a cell host.

"Promoter sequence" is a regulatory region of DNA capable of contact with RNA polymerase in a cell and initiating transcription of the coding sequence in the forward direction (3’). In accordance with the present invention this promotergene) it includes the minimum number of bases or elements necessary to initiate transcription at levels detectable above background levels. Within this promoter sequence is the site of transcription initiation, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. In most cases, but not always eukaryotic promoters contain "TA-TA"boxes and "SAT"boxes. In addition to the consensus sequences at -10 and -35 prokaryotic promoters contain sequences Shine-Dalgarno.

"Sequence expression regulation" is a DNA sequence that provides the control and regulation of transcription and translation of another DNA sequence. The coding sequence is under the control" of transcriptional and translational regulatory sequences in the cell in the case of RNA polymerase Transcriber the coding sequence into mRNA, which is then transmitted with the formation of the protein mass of 120 kDa, encoded this coding sequence.

"Signal sequence" can be present near the coding sequence tells the cell to the owner the ability to direct the polypeptide to the cell surface of cells or secrete it into the environment, this signal peptide is cleaved in the cell host, after which the protein mass of 120 kDa leaves the cell. The signal sequence may be present in combination with a variety of native proteins of prokaryotes and eukaryotes.

Used herein, the term "oligonucleotide" means a probe of the present invention and is a molecule consisting of two or more, and preferably from more than three deoxyribonucleotides. Its exact size depends on many factors, which, in turn, depend on the ultimate function and purpose of this oligonucleotide.

Used herein, the term "primer" means an oligonucleotide that regardless of whether there is it in nature in the form of a purified restriction digest or it was obtained synthetically, capable of acting as a site of initiation of synthesis when placed in conditions in which the induced synthesis of the product of the elongation of the primer, which is complementary to the nucleotide chain, i.e. in the presence of nucleotides and an inducing agent such as DNA polymerase and at suitable temperature and pH. This primer can be either single-stranded or he must be long enough to ensure that initsiirovat on many factors, including temperature, source of primer and the method of its use. For example, for diagnostic purposes, depending on the variability of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

Used herein, the primers are chosen so that they were substantially complementary to the various circuits of a specific target DNA sequence. This means that the primers must be sufficiently complementary so that they could gibridizatsiya with their respective circuits. So Primera sequence does not necessarily have to have a sequence identical to the sequence matrix. For example, complementary nucleotide fragment may be attached to the 5’-end of the primer, and the rest Primera sequence should be complementary to this circuit. Alternatively, complementary bases or longer sequences can be incorporated into the primer, provided that this Primera sequence has sufficient complementarity with these sequences or hybridizes with them, forming a t is s" and "restriction enzymes" refers to enzymes, each of which cut double-stranded DNA at specific nucleotide sequences or near it.

A cell is "transformed" by exogenous or heterologous DNA when introducing this DNA into the cell. Transforming DNA may or may not be integrated (covalently attached) into the genome of this cell. For example, in the cells of prokaryotes, yeast and mammals this transforming DNA may be located on episomal element, such as a plasmid. As for eukaryotic cells, a stably transformed cell is a cell in which the transforming DNA integrates into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones that make up the population of daughter cells containing the transforming DNA. "A clone is a population of cells derived from a single cell or its predecessor through mitosis. "Cell line" is a clone of the primary cell, which is capable of stable growth in vitro with the production of many generations.

Two sequences is available, at least about 75% (preferably at least about 80%, and most preferably at least about 90%, or 95%) according nucleotides. Sequences that are essentially homologous can be identified by comparing the sequences using standard software available in the data Bank sequences, or experiment by southern hybridization, for example, stringent conditions, as defined for that particular system. Appropriate hybridization conditions can be determined by each specialist. For example, Maniatis et al., see above; DNA Cloning, A Practical Approach", Vols. I & II, see above; "Nucleic Acid Hybridization", see above.

"Heterologous region" design DNA is an identifiable segment of DNA within a larger DNA molecule that is not found in nature in Association with this larger molecule. Thus, if the heterologous region encodes a gene of a mammal, the gene is usually flanked by DNA that does not flanks the genomic DNA of the mammal in the genome of the organism. In another example, the coding sequence is a construct where itself cadirci contains introns or synthetic sequence, having codons different from the native gene). Allelic diversity or natural mutations do not lead to the formation of heterologous DNA defined in the present description.

Marks, most often used in these studies are radioactive elements, enzymes, chemicals which fluoresce when irradiated with ultraviolet light, and others. A known number of fluorescent materials that can be used as labels. Such materials are, for example, fluorescein, rhodamine, auramine, Texas red, blue AMC and luciferase yellow. Specific detection material is anti-rabbit antibody produced in goat and conjugated to fluorescein by isothiocyanate.

Proteins can be tagged with a radioactive element or with an enzyme. Radioactive label can be detected by any existing methods of calculation. The preferred isotope may be selected from the3H,14C,32P,35S36Cl51Cr57Co.,58Co.,59Fe90Y125I131I and186Re.

Can also be used enzyme labels that can be detected luesch, amperometric or gasometrical methods. This enzyme kongugiruut with the selected particle by reaction with meticorten molecules, such as carbodiimides, diisocyanates, glutaraldehyde, etc., there are many enzymes that can be used in these procedures. The preferred are peroxidase,-glucuronidase,-D-glucosidase,-D-galactosidase, urease, glucose oxidase + peroxidase and alkaline phosphatase. Description of alternative materials and methods tagging can be found, for example, in U.S. patent No. 3654090, 3850752 and 4016043.

Professionals was developed and used specific analytical system, known as receptor analysis. In the receptor analysis of the analyzed material appropriately mark, and then a number of cellular test colony inoculant a certain number of the same label, and then conducting a study on the binding to determine the extent to which this labeled material bound to cellular receptors. This method can be determined differences in the affinity of these materials.

Specialists use analysis, known is th is usually a plasmid, continuously expressing need a specific receptor at its transfection into an appropriate cell line, and the second is a plasmid expressing a reporter such as luciferase, under the control of the receptor/ligand complex. For example, if you want to determine whether this compound is a ligand for a specific receptor, one of these plasmids should be designed so that it provides receptor expression in selected cell lines, and the second plasmid must contain a promoter that is attached to the luciferase gene, which was embedded reactive element for this specific receptor. If the test compound is an agonist for this receptor, the ligand will form a complex with the receptor and the resulting complex will be in contact with this reactive element and initiate transcription of the luciferase gene. Then the induced chemiluminescence was measured photometrically, build the curves of dependence "dose-response" and compare them with the curves obtained for known ligands. The above Protocol is described in detail in U.S. patent No. 4981784.

Used herein, the term "owner" means not only prokaryotes, but the encode immunoreactive protein mass of 120 kDa Ehrlichia canis present invention, can be used to transform a host using any method well known to every expert. For transformation of prokaryotes especially preferred is the use of a vector containing coding sequences of the gene encoding immunoreactive protein mass of 120 kDa Ehrllchia canis present invention.

Prokaryotic hosts may be E. coli, S. typhimurium, Serratia marcescenns, Mycobatrium vaccae and Bacillus subtllis. Eukaryotic hosts include yeast, such as Pichia pastoris, mammalian cells and insect cells.

Basically with this host are expressing vectors containing promoter sequences which facilitate the efficient transcription of the built-DNA fragment. Expressing the vector typically contains the site of initiation of replication, a promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. To achieve optimal cell growth of these transformed hosts can be fermented and cultured by methods known in the art.

The present invention relates mainly to clean the DNA that encodes an immunoreactive protein with a mass of 120 kDa Ehrlichia can what ina least of the 15 consecutive nucleotides (SEQ ID NO: 6). This protein mass of 120 kDa, encoded by the DNA of the present invention may have a sequence at least 80% identical (preferably 85%, more preferably 90%, and most preferably 95%) of the nucleic acid sequences listed in Fig.3 (SEQ ID NO: 7). More preferably, this DNA was included coding nucleotide sequence of SEQ ID NO: 8, or a degenerate variant of this sequence.

The probe, which hybridized DNA of the present invention, preferably consists of a sequence of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) the coding sequence of the nucleotides listed in SEQ ID NO: 8, or the complementary nucleotide. This probe can be used for detecting gene expression of immunoreactive protein with a mass of 120 kDa Ehrlichia canis in human cells by a method comprising the stage of: a) the interaction of the mRNA obtained from the cell with the labeled probe for hybridization; and (b) detecting hybridization of the probe with mRNA least of the 15 consecutive nucleotides, preferably 20, more preferably 30, even more preferably 50, and most preferably, all of the nucleotides) of the region consisting of nucleotides listed in SEQ ID NO: 8.

The term "high rigidity" refers to DNA hybridization and leaching conditions characterized by high temperature and low salt concentration, for example, to the conditions of leaching under 65When the salt concentration of approximately 0.1SSC or their functional equivalent. So, for example, conditions of high stringency can include hybridization at about 42In the presence of about 50% formamide; a first wash at about 65With about 2SSC containing 1% LTOs; and then the second washing of approximately 0.1SSC at about 65C.

"Basically pure DNA" means DNA that is not part of the environment, which usually is present in this DNA by separating the (partial or full clean-up) some or all of the molecules from the environment, or due to modifications of the sequences flanking this question DNA. is oudouse plasmid or virus, or in the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., cDNA or genomic DNA or cDNA fragment produced by polymerase chain reaction (PCR) or cleavage by the endonuclease) independent of other sequences. This term also means a recombinant DNA which is part of a hybrid gene encoding another paulilatino sequence, for example a hybrid protein. This term also means a recombinant DNA that includes a portion of the nucleotides listed in SEQ ID NO: 8, and which encode alternative slicing gene encoding immunoreactive protein mass of 120 kDa Ehrlichia canis.

This DNA can have a sequence that is at least about 70%, preferably at least 75% (e.g., at least 80%, and most preferably at least 90% identical to the coding sequence of the nucleotides listed in SEQ ID NO:8. The identity of these two sequences directly depends on the number of matches or identical provisions. If the position of the subunit in both of the two sequences employed similar Monomeric subunit, e.g., Engl. the AK, for example, if 7 positions in the sequence of 10 nucleotides identical to the corresponding provisions in the second 10-nucleotide sequence, these two sequences are identical at 70%. The length of the compared sequences in General is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. The sequence identity is usually defined using the software for sequence analysis (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705).

The present invention relates to a vector comprising a DNA sequence which encodes a gene that encodes an immunoreactive protein with a mass of 120 kDa Ehrlichia cants, and where the specified vector able to replicate in the host and includes at its right connections: a) the site of initiation of replication; b) a promoter; and C) a DNA sequence encoding this protein mass of 120 kDa. Preferably, the vector of the present invention contains the portion of the DNA sequence shown in SEQ ID NO: 8. "Vector" can be defined which can be used for amplification and/or expression of the nucleotide acid, coding immunoreactive protein mass of 120 kDa Ehrlichia canis. Expressing the vector is a replicable design, in which the nucleotide sequence encoding the polypeptide is correctly connected to a suitable regulatory sequence capable of expression of the polypeptide in the cell. Such regulatory sequences should be selected depending on the selected cells and the method of transformation. Mainly regulatory sequences are transcriptional promoter and/or enhancer, a suitable binding sites with mRNA and ribosomal sequences that regulate the termination of transcription and translation. To construct expressing vectors containing the appropriate signals in the regulation of transcription and translation, can be used by methods well known in the art. For example, the methods described by Sambruna and others, 1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, N. Y. Gene and its sequence transcription regulation are considered "properly connected" if these sequences regulating transcription effectively carry out the regulation of transcription of this gene. Vectors in h is Linyi viral vectors of the present invention are vectors, derived from retroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpes virus.

The term "mostly pure protein" means a protein that has been allocated, at least some of the components with which it is normally associated. Normally, this protein is mostly clean in that case, if he at least 60%, by weight does not contain proteins and other naturally occurring organic molecules with which it is normally associated in vivo. It is preferable that the purity of this preparation was at least 75%, more preferably at least 90% and most preferably at least 99% by weight. Mostly clean immunoreactive protein mass of 120 kDa Ehrlichia canis can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid that encodes an immunoreactive protein with a mass of 120 kDa Ehrlichia canis; or by chemical synthesis of the protein mass of 120 kDa. Purity may be determined using any suitable method, for example by column chromatography, such as immunoaffinity chromatography using antibodies specific for immunoreactive protein with a mass of 120 kDa Ehrlichia canis, electrophoresis in polyacrylamide in the case if he is separated from at least some of the impurities with which it is associated in nature. Thus, the protein was chemically synthesized or produced in a cellular system, which is not a cell, from which it inherently is, would be, by definition, protein, mainly not containing natural associated components. In accordance with this mostly pure proteins are eukaryotic proteins synthesized in E. coli and other prokaryotes or in any other organism in which they do not occur in nature.

In addition to basically a full-sized proteins of the present invention also relates to fragments (e.g., antigenic fragments) of immunoreactive protein with a mass of 120 kDa Ehrlichia canis (SEQ ID NO: 7). Used herein, the term "fragment" refers to a polypeptide, which usually has at least 10 residues, but mostly, at least 20 residues, and preferably at least 30 (e.g., 50) residues, but fewer residues than the intact sequence. Fragments immunoreactive protein with a mass of 120 kDa Ehrlichia canis can be generated by methods known in the art, for example by enzymatic geminata DNA using expressing vector, which encodes a defined fragment immunoreactive protein with a mass of 120 kDa Ehrlichia canis, or by chemical synthesis. The ability of the fragment candidate to show properties immunoreactive protein with a mass of 120 kDa Ehrlichia canis (for example, contact with the antibody-specific immunoreactive protein with a mass of 120 kDa Ehrlichia canis) can be evaluated by methods described here. Purified immunoreactive protein mass of 120 kDa Ehrlichia canis or antigenic fragments immunoreactive protein with a mass of 120 kDa Ehrlichia canis can be used to generate new antibodies or to test existing antibodies (for example, as a positive control in diagnostic analysis) in accordance with standard schemes known in the art. The present invention also includes polyclonal antisera produced using immunoreactive protein with a mass of 120 kDa Ehrlichia canis or fragment immunoreactive protein with a mass of 120 kDa Ehrlichia canis as an immunogen, for example, in rabbits. It uses standard protocols for the production of monoclonal and polyclonal antibodies, known to specialists. Monoclonal antibodies generated using this procedure can be scenester cDNA clones.

In addition, in the present invention includes a fragment immunoreactive protein with a mass of 120 kDa Ehrlichia canis, which, at least in part, encoded parts SEQ ID NO: 7, for example products of alternative mRNA splicing or alternative event processing protein, or in which the plot of this sequence has been delegated. A fragment of this protein or intact immunoreactive protein mass of 120 kDa Ehrlichia canis can be covalently linked to another polypeptide which is, for example, acts as a label, a ligand or agent that enhances antigenicity.

The term "pharmaceutically acceptable" refers to molecular particles and to compositions that, when administered to humans does not cause allergic or similar adverse reactions. Methods of obtaining water composition, which contains protein as an active ingredient is well known to specialists. Typically such compositions are prepared in the form of injection or in the form of liquid solutions or suspensions; this can be also obtained solid forms before injection can be dissolved or suspendresume in the liquid. This preparation can also be emulsified.

Protein can be prepared in the form of a composition in a neutral is f free amino groups of this protein and salt, formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, almond, etc., Salts formed free carboxyl groups can also be derived from inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron (3), and organic bases, such as Isopropylamine, trimethylamine, histidine, procaine, etc., After receipt of the compositions of the solutions imposed by the appropriate method for this pharmaceutical composition, and in such numbers, which is therapeutically effective. These compositions are administered in the form of various dosage forms such as solutions for injection.

For parenteral administration, for example, in aqueous solution, this solution should be suitably buffered if necessary and the liquid diluent first need to give isotonicity using sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In accordance with this MOU to the invention. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and can be either added to 1000 ml of liquid for subcutaneous injection or it can be entered in the site intended for infusion (for Example, "Remingtons Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). If necessary, the dose can be changed depending on the condition of the subject being treated. In any case, a suitable dose for each specific subject appoints specialist responsible for this treatment.

Specialists are well aware that this polypeptide may vary in its immunogenicity. Therefore, in most cases, it is necessary that the immunogen (e.g., the polypeptide of the present invention) was associated with a carrier. Preferred examples of such carriers are hemocyanin lymph snails (KLH) and bovine serum albumin human serum. Other carriers can be of various lymphokines and adjuvants, such as IL2, IL4, IL8, and others.

Means for conjugating the polypeptide to a protein carrier are well known in the art and they include glutaraldehyde, m-maleimidomethyl-N-hydroxysuccinimidyl, carbodiimide and bis-diazotized benzidine. It should also be noted that this peptide may bahororo know, the specific immunogenicity of the immunogen can be enhanced with the use of non-specific stimulators of the immune response, known as adjuvants. Preferred examples of adjuvants are complete adjuvant, BCG, Detox (RIBI, Immunochem Research Inc.), ISCOMS and aluminum hydroxide (Superphos, Biosector).

Vaccine preparations, which contain peptide sequences as active ingredients, basically well known in the art and illustrated in U.S. Patents№ 4608251; 4601903; 4599231; 4599230; 4596792 and 4578770, each of which is introduced into the present description by reference. Typically, such vaccines are manufactured in the form of injection or in the form of liquid solutions or suspensions; this can be also obtained solid forms before injection can be dissolved or suspended in a liquid. This preparation can also be emulsified. In most cases, the active immunogenic ingredient is mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable fillers are, for example, water, saline, dextrose, glycerol, ethanol and so forth, and combinations thereof. In addition, if necessary, the vaccine MSIE tabularasa agents or adjuvants, which increase the effectiveness of these vaccines.

Protein mass of 120 kDa, probably represents adhesin Ehrlichia spp. and differentially expressed on the cell surface of E. chaffeensis. Gene E. canis amplified by PCR using primers derived from the DNA sequences flanking the gene of E. chaffeensis. Gene E. canis cloned, sequenced and sverkhekspressiya in Escherichia coli. Protein mass of 120 kDa E. canis contains 14 tandem repeated units, each of which has a 36 amino acids. The DNA sequence of these repeats 94% homologous to each other. Complete amino acid sequence of the protein mass of 120 kDa E. canis 30% homologous protein with a mass of 120 kDa E. chaffeensis. The field of repetitions of proteins by mass of 120 kDa of these two species have a common amino acid sequence, which, as expected, are surface-exposed. Recombinant protein mass of 120 kDa E. canis reacted with serum obtained from convalescent dogs with arhelonom.

The present invention relates to gene, codereuse immunoreactive protein mass of 120 kDa Ehrlichia canis, and to the recombinant protein encoded by this gene. In one variation of its implementation of the present invention relates is but has the amino acid sequence of SEQ ID NO: 8, and the specified gene has the nucleotide sequence SEQ ID NO:7. In a preferred embodiment, the present invention relates to expressing the vector containing the gene encoding immunoreactive protein mass of 120 kDa Ehrlichia canis, and with the introduction of the vector into the cell he is able to Express this gene.

In another embodiment, its implementation of the present invention relates to a recombinant protein containing the amino acid sequence of SEQ ID NO:8. This amino acid sequence preferably encodes the nucleotide sequence of SEQ ID NO:7. The indicated recombinant protein contains 14 tandem repeated units, each of which has a 36 amino acids. More preferably, if the repeat units are hydrophilic. Even more preferably, if the specified recombinant protein is an antigen.

In a preferred embodiment, the present invention relates to a method for producing a recombinant protein with a mass of 120 kDa, comprising the stage of: obtaining a vector that contains expressing a region that includes the coding sequence of SEQ ID NO:8, is correctly connected to the promoter; transfection shirouma area.

Used herein, the term "complement" refers to the chain of nucleic acid, which hybridizes with the first nucleotide sequence by formation of double-stranded molecule under strict conditions. Stringent conditions are those conditions under which hybridization occurs between two nucleotide sequences having a high degree of homology, but does not occur hybridization of random sequences. So, for example, hybridization at low temperature and/or high ionic strength is defined as hybridization under low stringency, hybridization under high temperature and/or low ionic strength is defined as hybridization in conditions of high stringency. It should be noted that probes of a specific length, for sequences of a certain length and with a certain amount of reason and in the presence of formamide in the hybridization mixture is used, the temperature and ionic strength of a desired stringency.

Used herein, the term "engineered" or "recombinant" cell means a cell into which has been introduced recombinant gene, such as the gene encoding the antigen of E. chaffeensis. Therefore, engineered cells differ from that of natural cells, kotorii, having the gene or genes artificially introduced by man. Recombinante introduced genes are present either in the form of a cDNA of a gene or a genomic copy of the gene, or they represent the genes located near the promoter that is not normally associated with this introduced gene. In addition, this recombinant gene can be integrated into the host genome, or it may be present in the vector or in the bacterial genome, transfetsirovannyh in this cell the owner.

The following examples are provided to illustrate various embodiments of the invention and should not be construed as limiting the present invention.

Example 1

Erlichia

Strain Oklahoma Ehrlichia canis was provided by Dr. Jacqueline Dawson (Centers for Disease Control, Atlanta, GA). The Florida strain and three isolates of North Carolina E. canis, (Demon, DJ and Jake) were provided by Dr. Edward C. Breitschwerdt (College of Veterinary Medicine, North Carolina State University, Raleigh, NC). Strain Louisiana E. canis was provided by Dr. R. E. Corstvet (Louisiana State University, Baton Rouge, Louisiana). Ehrlichia were cultured in DH82 cells, macrophagecolony cell line dogs (J. E. Dawson, 1991). DH82 cells were collected with a scraper for cells in 100% infection erlichia. The cells were centrifuged at 17400g use the TT for 30 seconds on ice. The cell lysate was loaded onto a step gradient 42%-36%-30% renografin, and then was centrifuged at 80000g for 60 minutes. Erlichia in heavy and light bands were collected (Weiss E, 1975) and washed by centrifugation with Saharsa-phosphate-glutamate buffer (SPG, 218 mm sucrose, 3.8 mm KN2RHO4, 7.2 mm To2NRA4, 4.9 mm glutamate, pH 7.0).

Example 2

Obtaining DNA

Genomic DNA of Ehrlichia canis received from erlichia purified in the density gradient renografin using the kit for the extraction of nucleic acids IsoQuick (ORCA Research Inc., Bothell, WA) according to the manufacturer's instructions. Plasmid DNA was purified using the kit for plasmid isolation with a high degree of purity (Boehringer Mannheim Corp., Indianapolis, IN). The PCR product was purified using a kit PCR purification QIAquick PCR Purification Kit (QIAGEN Inc., Santa Clarita, CA).

Example 3

PCR amplification of the gene of the protein mass of 120 kDa E. canis

The primers were designed based on the DNA sequence of a gene of the protein mass of 120 kDa E. chaffeensis (SEQ ID NO:1-6, Fig.1) (Yu, X. J. 1996). The gene of the protein mass of 120 kDa E. canis amplified by PCR with 30 cycles: 94C, 30 s; 52With 1 min; and 72With 2 minutes PCR-amp is the DNA

DNA sequenced at the DNA sequencing machine (ABI Prism 377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA).

Example 5

Unidirectional deletion of the gene of the protein mass of 120 kDa E. canis

The scope of repetitions delegated from the 5’end of the gene of the protein mass of 120 kDa E. canis using partial hydrolysis restricteduse the endonuclease SpeI. Plasmid pCR120 first completely hydrolyzed by the enzyme XbaI, which had a unique cleavage site in this plasmid sequences near the 5’end of the gene of the protein mass of 120 kDa E. canis. Then, this plasmid is partially hydrolyzed by the enzyme SpeI. SpeI had a unique cleavage site in each repeat of the gene of the protein mass of 120 kDa E. canis, but had no website cleavage outside the scope of repetitions, including the sequence of the plasmid vector. To ensure appropriate representative partial hydrolysis from each mixture for hydrolysis took a sample every 5 minutes. The hydrolysis was stopped by adding EDTA to a final concentration of 50 mm and heating at 70C for 10 minutes.

After complete cleavage enzyme XbaI and partial cleavage enzyme SI removed (delegated) different number of repeating units, located between the XbaI site and each SpeI site R is the once). The mixture gidralizovanny restricteduse enzymes were processed by the fragment maple for completing the ends. Then the restriction mixture was separated by electrophoresis on an agarose gel to remove the plasmid of internal repeats, because the size of their molecules differed substantially. The mixture is delegated plasmids were extracted from the gel using a kit for the extraction of the gel (QIAquick Gel Extraction Kit (QIAGEN Inc., Santa Clarita) and subjected recircularisation using T4 ligase. These delegated plasmids were ransformational in strain DH5E. coli and selected for sequencing in accordance with their sizes. An alternative area of repeats of the gene of the protein mass of 120 kDa E. canis unilaterally delegated with the 3’-end using ectonucleoside III and system Erase-a-Base (Promega Corp., Madison, WI) according to manufacturer's instructions.

Example 6

Determining the number of repeats in the gene of the protein mass of 120 kDa E. Canis

PCR amplificatory gene protein mass of 120 kDa E. canis from all E. canis cloned in a cloning vector pCR2.1 TA (Invitrogen). The recombinant plasmid was first digested with the enzyme EcoRI, and then partially digested with the enzyme SpeI, as described above. Mixture for cleavage were separated by electrophoresis and hybridized with the oligonucleotide probe, derived from the sequence above from the field repeats of the gene of the protein mass of 120 kDa E. canis. The DNA probes were labeled using digoxigenin-11-dUTP using the kit to attach to DIG oligonucleotide (Boehringer Mannhiem Co., Indianapolis, In) according to the manufacturers Protocol.

Example 7

Analysis of genes

The DNA sequence and deduced amino acid sequences were analyzed using the software of the Wisconsin GCG (Genetic Computer Group, Inc., Madison, WI) and the software DNASTAR (DNASTAR, Inc., Madison, WI). The signal sequence of the extracted protein by mass of 120 kDa were analyzed using the program PSORT (World Wide Web site at URL: pttp://psort. nibb. ac. jp), which allows using methods McGeoch (D. J. McGeoch, Virus Research, 3, 271, 1985) and Von Heijne (von Heijne G. 1986, Nucl.Acids Res., 14, 4683) to suggest the presence of signal sequences, and using the method of Klein and others (P. Klein, M. Kanehisa & C. DeLisi, Biochem. Biophys. Acta, 815, 468, 1985) to detect possible transmembrane domains.

Example 8

Expression of the gene of the protein mass of 120 kDa E. canis in E. coli

Direct cloning of the gene of the protein mass of 120 kDa E. canis in expressing vector pGEX (Amersham Pharmacia Biotech, Piscataway, NJ) was not due to the lack of appropriate sites splitting restrictio the simulation vector pGEX. The coding region of the gene of the protein mass of 120 kDa E. canis from nucleotides 175 to nucleotide 1793 amplified by PCR using the direct primer (SEQ ID NO:13) and reverse primer (SEQ ID NO:14). PCR products were cloned into the cloning vector pCR2.1 TA (Invitrogen) with the creation of the EcoRI restriction site at both ends of the insert. The insert in the recombinant plasmid pCR2.1 cut EcoRI and was isolated from the plasmid DNA in agarose gel. This insert was extracted from agarose gel using a kit for the extraction of the gel (QIAquick Gel Extraction Kit (QIAGEN Inc., Santa Clarita) and cloned into EcoRI-hydrolyzed vector pGEX. Protein of E. canis expressed in the BL21 strain of E. coli as GST-hybrid protein. This GST-hybrid protein mass of 120 kDa was subjected to affinity purification using granules of glutathione-sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ). Recombinant protein mass of 120 kDa E. canis was tsalala from GST-hybrid protein thrombin.

Example 9

Immunization of mice

Mice were immunized with recombinant protein with a mass of 120 kDa E. canis to produce antisera. Recombinant protein-R120 was mixed with an equal volume of complete adjuvant's adjuvant for the first injection and incomplete adjuvant's adjuvant for subsequent injections. Mice were immunized NutriBar

Detection of the gene of the protein mass of 120 kDa E. canis

In an attempt detection of the gene of the protein mass of 120 kDa E. canis used southern blotting. 1,2 -, etc., called DNA fragment, amplificatory of the gene of the protein mass of 120 kDa E. chaffeensis by PCR using pairs of primers pxcf3b (SEQ ID NO:3) and pxar4 (SEQ ID NO:5, Fig. 1), were marked by digoxigenin-11-dUTP and used as a probe for the detection of homologous gene in E. canis by southern blotting. Southern blotting showed that the probe for the gene of the protein mass of 120 kDa E. chaffeensis was not hybridisable with EcoRI-hydrolyzed genomic DNA of E. canis in the conditions under which this probe gave a strong hybridization with the genomic DNA of E. chaffeensis. This result indicates that the gene of the protein mass of 120 kDa E. canis is significantly different from the homologous gene of the protein mass of 120 kDa E. chaffeensis.

Although the similarity between the genes of the protein mass of 120 kDa E. canis and E. chaffeensis is low, however, it is assumed that they may contain some conservative domains, which can be used to design PCR primers suitable for amplification of both genes of the protein mass of 120 kDa. Therefore, this homologous gene of the protein mass of 120 kDa in E. canis (strain Oklahoma) was further amplified by PCR. The primers derived from g is of generowania gene protein mass of 120 kDa E. chaffeensis (Fig.1). To construct nine primer pairs were used three forward primers (SEQ ID NO:1-3), each of which was 3 couples with reverse primers (SEQ ID nos:4-6). PCR results demonstrated that the DNA of E. canis was not amplified using primers within the coding region of the gene of the protein mass of 120 kDa E. chaffeensis. 2,5, etc., called DNA fragment amplified from genomic DNA of E. canis using a pair of primers pxcf2-2 and pxar3, derived from non-coding DNA sequences flanking the gene of the protein mass of 120 kDa E. chaffeensis (Fig.1).

Example 11

Determination of the number of repeating units in the gene of the protein mass of 120 kDa E. canis

To illustrate the number of repeating units, located in the gene of the protein mass of 120 kDa E. canis, used southern blotting. Restriction analysis of the DNA sequence of a gene of the protein mass of 120 kDa strain Oklahoma showed that all retries have a unique site of cleavage by the endonuclease SpeI. The insert in the plasmid pCR120 was partially hydrolyzed with SpeI. As a result of incomplete hydrolysis of the DNA of the gene of the protein mass of 120 kDa E. canis enzyme SpeI was formed three kinds of DNA fragments: repetitions with a 5’-terminal non-repeating sequence, internal repeats and repeats to the 3’end is not what audace gene sequence was started from the same position (5’end of the gene), but ended at different times. These DNA fragments were isolated by agarose gel according to their length, which corresponded to the number of repetitions. DNA was transferred to nylon membrane and used for hybridization with DIG-labeled oligonucleotide. This oligonucleotide (SEQ ID NO:15) was derived from the DNA sequence from nucleotide 38 to nucleotide 59, which were located above the area of repetitions. So this oligonucleotide was hybridisable only DNA fragments, which were 5’-end of this gene, but not with internal repeats and not with reruns from the 3’end of the gene. Thus, the number of bands detected using an oligonucleotide probe that corresponds to the number of repetitions. Southern blotting revealed a ladder of 14 bands with an increment of 108 p. N. in E. canis (Fig.2). The results showed that E. canis is present in 14 iterations with 108 p. N. each.

Example 12

Analysis of the DNA sequence of a gene of the protein mass of 120 kDa E. canis

PCR amplificatory the DNA fragment of the E. canis cloned in the vector pCR2.1. The obtained recombinant plasmid was designated RSA. This DNA plasmids RSA used as template for sequencing of the insert DNA of E. canis. First, the DNA sequence was obtained with the use of the insert. Subsequent DNA sequencing was performed by "primerno walk" insert and unidirectional deletions insert in the plasmid RSA by restriction-endonuclease or exonuclease III. Sequence analysis of the DNA showed that the insert DNA contained an open reading frame (ORF) consisting of 2084 nucleotides (SEQ ID NO:7), which encodes 688 amino acids (SEQ ID NO:8). This open reading frame was identified as the gene of the protein mass of 120 kDa E. canis. Any consensus DNA sequence of the promoter of E. coli, which is located near the 5’end of this gene was not detected. N-end of the deduced amino acids did not coincide with the consensus sequence of the signal peptide of E. coli. In the gene of the protein mass of 120 kDa E. canis was attended by 14 tandem repeats. Each repetition consisted of 108 nucleotides and encodes a 36 amino acids (Fig.3). Homology of amino acid sequences of all retries exceeded 94% (Fig.4). Before the first repeat was present incomplete repeat, which had a deletion of 7 amino acids (Fig.3) and had a 70% homology with other repetitions.

Using the program FastA search in Genbank, it was found that the gene of the protein mass of 120 kDa E. canis has no significant homolog is Yes it Is. canis (SEQ ID NO: 10) and E. chaffeensis (SEQ ID NO:9) 30% (Fig.5). Proteins with a mass of 120 kDa are more conservative at its N end and repeats. Amino acid homology to the first 32 amino acids at the N-end of the protein mass of 120 kDa E. canis and E. chaffeensis is 50%. Homology of the DNA sequence of genes for proteins with a mass of 120 kDa of these two species is 58%. Was sequenced non-coding DNA sequence upstream 340 p. N. gene protein mass of 120 kDa E. canis, and it was found that these non-coding region adjacent to the genes of proteins by mass of 120 kDa of these two species of Ehrlichia, have 84% homology.

Example 13

Predicted localization and antigenicity of the protein mass of 120 kDa E. canis

Deduced amino acid sequence of the gene of the protein mass of 120 kDa E. canis was analyzed on the hydrophobicity, the probability of surface exposure and antigenicity using "Protein" software Lasergene (DNASTRA Inc., Madison, WI). It is assumed that all of the recurring units are hydrophilic and exhibited on the surface (Fig.6). Comparison of proteins of E. canis and E. chaffeensis demonstrated that all repeating units in both proteins are surface-exposed. These poverhnosti suggest, the protein mass of 120 kDa E. canis is the outer membrane protein. Hydrophilic amino acids in these repeats may represent surface-exposed part of this protein.

Analysis of proteins was performed by the method of the jameson-wolf, which allowed us to predict the possible antigenic determinants showed that proteins with a mass of 120 kDa as E. canis, chaffeensis are likely to be highly antigenic (Fig.6). Analysis of proteins was performed by the method Roth-bard-Taylor, who has revealed a possible localization of antigenic determinants of T-lymphocytes, demonstrated that a protein mass of 120 kDa E. canis has several suspected T-cell epitopes and that they are all localized on the sequences located outside the domain of repetitions. In contrast, each repeat protein with a mass of 120 kDa E. chaffeensis had two T-cell epitope.

Example 14

Homologous genes in other strains of E. canis

For amplification of the gene of the protein mass of 120 kDa from other strains of E. canis was using PCR. 2,5, etc. N. a fragment of DNA from all strains of E. canis, including strains Florida, Louisiana and three dog isolate New Carolina: Demon, DJ and Jake amplified using primers PXCf2-2 and Rhag. The gene segments of the protein mass 120 to the DNA sequence, above and below from the field duplicates, were identical for all strains of E. canis. The full scope of repetitions for all strains of E. canis was not sequenced because for sequencing it is necessary that this gene has been delegated. Was sequenced only the last repetition for all strains, and the first repeat to strain DJ. Sequence first repeat strains DJ and Oklahoma were identical. The last sequence of the repeat were identical for all strains. The homology of the gene of the protein mass of 120 kDa from all strains of E. canis, was also demonstrated identical SI-physical restriction maps (Fig.8).

Example 15

Electrophoresis in SDS page with LTOs and Western blot analysis

The gene of the protein mass of 120 kDa E. canis was sverkhekspressiya in E. coli (Fig.9). Recombinant protein mass of 120 kDa, encoded 1620 p. H. DNA fragment that includes the full scope of repeats of the gene of the protein mass of 120 kDa, were produced as GST-hybrid protein. The molecular weight of the hybrid protein mass of 120 kDa, estimated on LTO-gel was approximately 140 kDa and significantly exceeded the expected molecular mass of the full-size protein mass of 120 kDa E. canis, which was only 73,6 kDa and was predicted on the basis of and the th 120 kDa reacted with a protein with a mass of 120 kDa E. canis (Fig.10). Dog anticavity against E. canis also reacted with the recombinant protein with a mass of 120 kDa E. canis (Fig.11).

Example 16

Discussion

Although Ehrlichia spp. are obligate intracellular bacteria, however, neither gene adhesin or gene invasin Herlihy not been identified. Was previously cloned and sequenced the gene of the protein mass of 120 kDa E. chaffeensis (Yu, 1997). Recently it was demonstrated that a protein mass of 120 kDa E. chaffeensis is an outer membrane protein that is expressed predominantly on plotnikovo ultrastructural form E. chaffeensis, but not reticular cells. Non-invasive non-adhesive strain of E. coli expressing the protein mass of 120 kDa, was developed capacity for adhesion and penetration in cultured mammalian cells. These results suggest that protein mass of 120 kDa should be adhesion or invasion, and therefore it can be considered as a candidate for the vaccine.

Ehrlichia canis and E. chaffeensis in their genetic and antigenic properties are closely related bacteria. Homology between E. canis and E. chaffeensis for gene 163 rRNA is 98% and for nadA gene (Yu, 1997) - 89%. Since it is obvious that a protein mass of 120 kDa plays an important role in the accession is e, in E. canis may be similar gene protein mass of 120 kDa E. chaffeensis, which may have the same biological function. This assumption was confirmed by PCR amplification of the gene of the protein mass of 120 kDa E. canis using primers derived from the gene of E. chaffeensis. However, the DNA of the gene of the protein mass of 120 kDa E. canis has no significant homology with the homologous gene of E. chaffeensis. Homology of the genes of the protein mass of 120 kDa E. canis and E. chaffeensis is only 30%. The low homology of the genes of these two species of Ehrlichia may explain the absence of a southern blot hybridization of DNA E. canis with a probe for the gene of the protein mass of 120 kDa E. chaffeensis and the absence of PCR amplification of the gene of E. canis using primers derived from the coding region of the gene of the protein mass of 120 kDa E. chaffeensis. It was unexpectedly found that non-coding sequences flanking genes of the protein mass of 120 kDa, are more conservative than the coding sequences of genes of the protein mass of 120 kDa E. canis and E. chaffeensis. From an evolutionary point of view, it is expected that the coding sequence, which is under selection pressure is more conservative than non-coding sequence, in which the mutation should not be expected to affect the however, obviously, what gene E. canis is homologous to the gene of the protein mass of 120 kDa E. chaffeensis, which gives reason to believe that they are localized in the same positions in the respective genome and they have 30% homology; in particular, they have a common motifs in the repeat region. However, both the gene of the protein mass of 120 kDa E. canis and E. chaffeensis have different amino acid sequences and the number of repeats, a protein mass of 120 kDa E. chaffeensis contains 4 repetitions, each of which consists of 80 amino acids and a protein mass of 120 kDa E. canis has 14 repeats, each of which consists of 36 amino acids. However, these repetitions in both proteins are hydrophilic and assumes that they are surface-exposed. Despite differences in the number of iterations, even the total number of surface-exposed regions in these repetitions of these two proteins is very similar. The repeat units of both proteins share a common motif consisting of identical amino acids that are hydrophilic and form the core (cor) surface-exposed domains of these proteins. The repeat units of both proteins rich in serine and glutamic acid. And serine and glutamic acid, each accounts for 19% of all amino acids of the unit E. chaffeensis, respectively. While using the program for the intended protein localization PSORT signal sequence was not detected at the N end of the deduced amino acid protein with a mass of 120 kDa E. canis, but it is clear that a protein mass of 120 kDa E. canis is a surface protein similar to protein mass of 120 kDa E. chaffeensis, in which the signal sequence is also absent (Yu, 1997).

Similarly, the gene of the protein mass of 120 kDa E. chaffeensis predicted molecular mass of the protein mass of 120 kDa E. canis significantly exceeds the size of the molecule, determined by the electrophoretic mobility of the protein in SDS page-ordinator. A similar phenomenon has been described for other proteins containing domains of repetitions, including proteins of Anaplasma marginale (Allred D. R. et al., 1990), Plasmodium (D. J. Kemp, 1987), Staphylococcus aureus (Hollingshead 1986, Signas 1989), and 100-kDa and 130-kDa proteins of granulocyte Ehrlichia (HGE) man (J. R. Storey, 1998). Repeating units of 100-kDa and 130-kDa proteins HGE have a common sequence with the protein mass of 120 kDa E. chaffeensis (Storey, 1998). Amaranta migration of proteins by mass of 120 kDa E. canis and E. chaffeensis is not a consequence of the high percentage content of some amino acids, because the molecular mass of this protein by mass of 120 kDa higher than the molecular mass predskazanny the addition of the protein mass of 120 kDa, such as glycosylation. Posttranslational modification of proteins by mass of 120 kDa E. canis and E. chaffeensis is currently under study. Because protein mass of 120 kDa E. chaffeensis differentially expressed in various structural forms of E. chaffeensis, the protein mass of 120 kDa may play a role in the pathogenesis of infection with E. chaffeensis. In the present research in order to find out whether expressed protein mass of 120 kDa E. canis mainly in plotnikovo cell of E. canis. Although the gene of the protein mass of 120 kDa of most strains of E. canis is not completely sequenced, but based on the fact that a known sequence that does not include field duplicates, as well as the first and last iterations are identical to strains of E. Canis, and based on the fact that all strains of E. canis have the same number of iterations, we can assume that the sequence of the gene of the protein mass of 120 kDa identical for all strains of E. canis. A high degree of homology to the DNA sequence and is identical to the number of repeats of the gene of the protein mass of 120 kDa in all strains of E. canis suggests that the strains of E. canis have less genetic diversity than the strains of E. chaffeensis in which the number of repeats in the gene for protein masem in the prior art, belongs to the present invention. These patents and publications are introduced by reference as if each individual publication was specifically and individually introduced by reference.

For every person it is obvious that the present invention can be suitably adapted to achieve the above aims and advantages inherent in the present invention. The above examples, methods, procedures, treatments, molecules, and specific compounds described in the present application, are preferred embodiments of the invention and are illustrative, and therefore they should not be construed as limiting the scope of the invention. In the present invention can be introduced modifications and other uses of the present invention, does not extend, however, beyond being and scope of the invention stated in the claims.

Sources of information

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3. Anderson, B. E., et al., 1992. Int. J. Syst. Bacteriol. 42:299-302.

4. Bakken, J. S., et al., 1994. JAMA. 272:212-8.

5. Buhles, W. C. Jr., et al., 1974. J. Infect. Dis 130:357-367.

6. Chen, S. M., et al., 1994. J. Clin. Environ. 32:589-95.

7. Ativan.E., et al., 1984. Canine ehrlichiosis. p. 545-561. In C. E. Greene (ed). Clinical microbiology and infectious diseases of the dog and cat. The W. B. Saunders Co., Philadelphia.

11. Groves, M. G., et al., 1975. Am. J. Veterinary Research. 36:937-40.

12. Hollingshead, S. K., et aL, 1986. J. Biol. Chem. 261:1677-86.

13. Kemp, D. J., et al., 1987. Annu. Rev. Environ. 41:181-208.

14. Jameson, B. A., et al., 1988. CABIO, 4:181-186.

15. KIein, P., et al., 1985. Biochem. Biophys. Acta, 815: 468-76.

16. Lewis, G. E. Jr., et al., 1977. American Journal of Veterinary Research. 38:1953-5.

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Claims

1. The selected segment of nucleic acid that encodes a protein mass of 120 kDa Ehrlichia canis, where this protein is immunoreactive with respect to anticigarette against Ehrlichia canis, and where this protein has the amino acid sequence of SEQ ID NO: 8.

2. The selected segment of nucleic acid on p. 1, characterized in that the specified segment has the nucleotide sequence SEQ ID NO: 7.

3. Expressing plasmid vector containing CE is epted, encoded by SEQ ID NO: 7.

4. Recombinant protein mass of 120 kDa, containing the amino acid sequence of SEQ ID NO: 8.

5. Recombinant protein under item 4, characterized in that the amino acid sequence encoded by a nucleic acid segment containing the sequence of SEQ ID NO: 7.

6. Recombinant protein under item 5, characterized in that the segment of the nucleic acid contained in expressing vector.

7. Recombinant protein under item 4, characterized in that it contains 14 tandem repeated units, each of which consists of 36 amino acids.

8. Recombinant protein under item 7, characterized in that the said repeating units are hydrophilic.

9. Recombinant protein under item 4, characterized in that it is an antigen.

10. Antibody immunoreactive with respect to a protein with the amino acid sequence SEQ ID NO: 8, obtained by immunization of a mammal specified protein.

11. Method of inhibiting infection caused by Ehrlichia canis, the entity providing the identification stage of the subject, presumably exposed to or infected with Ehrlichia canis; and introducing the composition containing the antigen mass of 120 kDa Ehrlichia canis, koudstaal a recombinant protein, containing the amino acid sequence of SEQ ID NO: 8.

12. The method according to p. 11, where the indicated recombinant protein mass of 120 kDa and is encoded by gene containing the sequence of SEQ ID NO: 7.

 

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