RU2237716C2 - Dna sequence encoding ehrlichia canis protein with molecular mass 30 kda, vector, recombinant protein and method for it preparing - Google Patents

Abstract

FIELD: molecular biology, genetic engineering.

SUBSTANCE: invention proposes DNA sequence encoding Ehrlichia canis protein with molecular mass 30 kDa. Also, invention proposes vector for preparing protein comprising DNA sequence, recombinant protein for preparing antibodies taken among the group consisting of SEQ ID NO:4 and 6, and a method for it preparing. Recombinant protein from Ehrlichia canis with molecular mass 30 kDa shows immune reactivity to serum raised against Ehrlichia canis. Proposed group of inventions can be used for the development of vaccine against canine ehrlichiosis.

EFFECT: improved preparing method, valuable properties of protein.

12 cl, 10 dwg, 18 ex

Description

BACKGROUND OF THE INVENTION

Field of Invention

The present invention relates generally to the field of molecular biology. More specifically, the present invention relates to molecular cloning and characterization of genes encoding homologous proteins with a molecular weight of 28 kDa in Ehrlichia canis and a multigenic locus encoding homologous proteins with a mass of 28 kDa in Ehrlichia canis, as well as their use.

Description of the prior art

Dog ehrlichiosis, also known as tropical dog pancytopenia, is a tick-borne rickettsiosis of domestic dogs, first described in Africa in 1935 and in the United States in 1963 (Donatien & Lestoquard, 1935; Ewing, 1963). The disease became better diagnosed after an epizootic outbreak that occurred in US Army dogs during the Vietnam War (Walker et al., 1970).

The causative agent of dog ehrlichiosis is Ehrlichia canis, a small gram-negative obligate intracellular bacterium that exhibits tropism for mononuclear phagocytes (Nyindo et al., 1971) and is transmitted by the common dog tick Rhipicephalus sanguineus (Groves et al., 1975). The development of dog ehrlichiosis is divided into three stages - acute, subclinical and chronic. The acute stage is characterized by fever, anorexia, depression, lymphadenopathy, and mild thrombocytopenia (Troy & Forrester, 1990). Typically, dogs recover from an acute stage, but become permanently infected with carriers of the pathogen without clinical symptoms of the disease for months and even years (Harrus et al., 1998). The chronic stage, which develops in some cases, is characterized by thrombocytopenia, hyperglobulinemia, anorexia, exhaustion and bleeding, in particular nosebleeds, with subsequent death (Troy & Forrester, 1990).

Molecular taxonomic analysis based on the parameters of the 163 rRNA gene determined that E.canis and E. chaffeensis, the causative agent of human monocytic ehrlichiosis (MEX), are closely related (Anderson et al., 1991; Anderson et al., 1992; Dawson et al., 1991; Chen et al., 1994). Significant cross-reactivity of antigens with molecular weights of 64, 47, 40, 30, 29, and 23 kDa was reported in E.canis and E. chaffeensis (Chen et al., 1994; Chen et al., 1997; Rikihisa et al., 1994 ; Rikihisa et al., 1992). An immunoblot analysis of immunoreactive antigens from human and dog sera taken during the recovery period allowed the identification of numerous immunodominant E.canis proteins, including a protein with a molecular weight of 30 kDa (Chen et al., 1997). In addition, a protein of 30 kDa E.canis was described as the main immunodominant antigen recognized by the early immune response, which differs in antigenic properties from a protein of 30 kDa E. chaffeensis (Rikihisa et al., 1992; Rikihisa et al., 1994) . Other immunodominant E.canis proteins with molecular weights in the range of 20-30 kDa have also been identified (Brouqui et al., 1992; Nyindo et al., 1991; Chen et al., 1994; Chen et al., 1997).

Recently, the cloning and sequencing of a multi-gene family (ocm-1) encoding 23-28 kDa proteins has been described in E. chaffeensis (Ohashi et al., 1998). A 28 kDa (p28) outer membrane immunodominant gene protein was cloned in E. chaffeensis homologous to the map-1 gene of Cowdria ruminantium. Mice immunized with the recombinant P28 protein were protected against infection with a homologous strain according to the results of PCR analysis of peripheral blood 5 days after infection (Ohashi et al., 1998). Molecular cloning of two similar but non-identical tandemly organized protein genes with a mass of 28 kDa in E.canis homologous to the mpr-1 family of E. chaffeensis and the marm-1 gene of C. ruminantium (Reddy et al., 1998) was also reported.

The prior art is characterized by the lack of data on the cloning and characterization of new genes of homologous immunoreactive proteins with a mass of 28 kDa in Ehrlichia canis and a single multigenic locus including homologous genes of proteins with a mass of 28 kDa. In addition, in the prior art there is no data on recombinant proteins encoded by such immunoreactive genes in Ehrlichia canis. The present invention fills this long-standing gap and an urgent need in the art.

SUMMARY OF THE INVENTION

The present invention describes the molecular cloning, sequencing, characterization and expression of genes of homologous mature immunoreactive proteins with a mass of 28 kDa in Ehrlichia canis (designated Esa 28-1, ECa28SA3 and ECa28SA2), as well as the identification of a single locus (5592 nucleotides (bp)), including five protein genes weighing 28 kDa of Ehrlichia canis (ECa28SAl, ECa28SA2, ECa28SA3, Eca28-1 and ECa28-2). Comparisons with E. chaffeensis and between 28 kDa E.canis protein genes showed that EC-28-1 shows the highest level of amino acid sequence homology with the multi-gene family omp-1 E. chaffeensis and is highly conserved among E.canis isolates. Five 28 kDa proteins are predicted to include signal segments, cleavage of which gives mature proteins, and the level of amino acid homology is in the range of 51-72%. Analysis of intergenic spacers revealed hypothetical promoter regions for each gene: this confirms that these genes can be expressed independently and differentially. The size of non-coding intergenic spacers is from 299 to 355 bp, and they are 48-71% homologous.

In one embodiment, the present invention relates to DNA sequences encoding an Ehrlichia canis 30 kDa immunoreactive protein. Preferably, the protein has an amino acid sequence selected from the group that includes SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and the gene has a nucleotide sequence selected from the group that includes SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, and is part of a polymorphic multigenic family. Typically, a protein includes an N-terminal signal sequence that is cleaved during post-translational processing, resulting in a mature protein of 28 kDa mass. Even more preferably, DNAs encoding proteins of 28 kDa are part of a single multigenic locus, the size of which is 5592 bp and which encodes all five homologous proteins with a mass of 28 kDa in Ehrlichia canis.

In another embodiment, the present invention relates to an expression vector comprising a gene encoding an Ehrlichia canis 28 kDa immunoreactive protein capable of expressing a gene by introducing a vector into the cell.

In yet another embodiment, the present invention relates to a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group which includes SEQ ID NO: I, SEQ ID NO: 3 and SEQ ID NO: 5. Preferably, the recombinant protein includes four variable located on the cell surface of the site, which are hydrophilic and antigenic. Recombinant protein can be used as an antigen.

In yet another embodiment, the present invention relates to a method for producing a recombinant protein, comprising the steps of preparing a vector that includes an expression region comprising a sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 operably linked to the promoter; cell transfection with a vector; and culturing the cell under conditions effective for expression of the expression site.

Also, the present invention can be described in some embodiments as a method of suppressing an Ehrlichia canis infection in a subject, the method comprising the steps of: identifying a subject for whom an exposure or infection of the Ehrlichia canis is suspected; and administering a composition containing a 28 kDa antigen of Ehrlichia canis in an amount effective to suppress Ehrlichia canis infection. Suppression can occur by any means, such as, for example, stimulation of a humoral or cellular immune response in a subject, or by other means, such as suppressing the normal function of an antigen weighing 28 kDa, or even competing with an antigen for interaction with a certain agent in the subject's body.

Other and subsequent aspects, properties, and advantages of the present invention will become apparent from the following description of the preferred embodiments of the invention considered, given in connection with the purpose of its disclosure.

Brief Description of the Drawings

Given that a material that understands the above properties, advantages and objects of the present invention, as well as another that becomes clear, is set forth and can be understood in detail, a more detailed description of the invention, a brief summary of which was given above, may be made by reference to some of its implementation, which are illustrated in the accompanying drawings. These drawings form part of this application. It should be noted, however, that the accompanying drawings illustrate preferred embodiments of the present invention and, therefore, are not intended to limit its scope.

Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) and the decoded amino acid sequence (SEQ ID NO: 2) of the EC-28-1 gene, including flanking 5'- and 3'-non-coding sequences. The ATG start codon and TAA stop codon are in bold, and the 23 amino acid leader signal sequence is underlined.

The figure 2 shows the SDS-PAGE electrophoresis of the expressed recombinant chimeric protein EC-28-1-thioredoxin with a molecular weight of 50 kDa (lane 1, arrow) and a control thioredoxin with a mass of 16 kDa (lane 2, arrow), as well as the corresponding immunoblot of the recombinant chimeric protein EC-28-1-thioredoxin recognized by a dog antiserum that is recovering from infection with E.canis (lane 3). The control thioredoxin antiserum to E.canis was not detected (not shown).

The figure 3 shows a comparison of the amino acid sequences of the protein EC-28-1 (SEQ ID NO: 2) and EC-28SA2 (partial sequence: SEQ ID NO: 7) and EC-28SAl (SEQ ID NO: 8), P28 E.chaffeensis (SEQ ID NO: 9 ), OMP-1 family of E. chaffeensis (SEQ ID NO: 10-14) and C. ruminantium MAP-1 (SEQ ID NO: 15). The amino acid sequence of EC-28-1 is presented as a consensus sequence. The amino acids identical to the amino acids EC-28-1 are not shown - they are given by dots. Differing amino acids are indicated by their corresponding single letter designations. Gaps made to ensure maximum amino acid sequence matching are shown by dashes. Variable segments are underlined and labeled (VR1, VR2, VR3 and VR4). Arrows indicate the putative signal of the peptidase cleavage site of the signal peptide.

Figure 4 shows the phylogenetic relationships of EC-28-1 with EC-28SA2 (partial sequence) and ECa28SAl of E.canis, six members of the omp-1 family of E. chaffeensis omp-1 and C. ruminantium mar-1 from decrypted amino acid sequences using an unbalanced tree construct. The length of each pair of branches corresponds to the distance between the amino acid sequences in pairs. The scale bar determines the distance between sequences.

Figure 5 shows Southern blotting data of E.canis genomic DNA, completely digested with six separate restriction enzymes and hybridized to a digoxigenin-labeled EC-28-1 probe (lanes 2-7); Digoxigenin-labeled molecular weight markers (lanes 1 and 8).

The figure 6 shows a comparison of the characteristics of the predicted proteins EC-28-1 (strain Jake) and P28 E.chaffeensis (strain Arkansas). Surface probability predicts surface residues using a hexapeptide window. The surface residue is a residue with an excess of 2.0 nm ² over the surface area available to water. Hexapeptide, characterized by a value of more than 1, was considered as a surface area. Antigenic index predicts potential antigenic determinants. Sites characterized by values above 0 are potential antigenic determinants. A T cell motif localizes potential T cell antigenic determinants using a 5 amino acid motif: residue 1 is glycine or polar, residue 2 is hydrophobic, residue 3 is hydrophobic, residue 4 is hydrophobic or proline, and residue 5 is polar or glycine. The line indicates the position of amino acids.

The figure 7 shows the nucleic acid sequence and the decoded amino acid sequence of the genes of proteins with a mass of 28 kDa E.canis - ECa28SA2 (nucleotides 1-849: SEQ ID NO: 3; amino acid sequence: SEQ ID NO: 4) and ECa28SA3 (nucleotides 1195-2031: SEQ ID NO: 5; amino acid sequence of SEQ ID NO: 6), including intergenic non-coding sequences (NC2, nucleotides 850-1194: SEQ ID NO: 31). The ATG start codon and stop codons are in bold.

Figure 8 schematically shows five genes of a 28 kDa protein locus with E.canis mass (5592 bp), which indicates the genomic orientation and non-coding intergenic regions (28NC1-4). The 28 kDa protein genes shown at locus 1 and 2 (shaded) have been described previously (McBride et al., 1999; Reddy et al., 1998; Ohashi et al., 1998). The complete sequence of ECaSA2 and the new protein gene with a mass of 28 kDa designated ECa28SA3 (without shading) was determined. Non-coding intergenic regions (28NC2-3) between ECaSA2, ECa28SA3 and ECa28-1 were completely determined by connecting previously unbound loci 1 and 2.

Figure 9 shows the phylogenetic relationships of five protein genes weighing 28 kDa E.canis in terms of amino acid sequence parameters using the construction of an unbalanced tree. The length of each pair of branches corresponds to the distance between pairs of amino acids. The scale bar determines the distance between sequences.

Figure 10 shows a comparison of nucleic acid sequences of a non-coding intergenic spacer of protein genes weighing 28 kDa E.canis (SEQ ID NO: 30-33). Unshown nucleic acids indicated by a dot (.) Are identical to non-coding region 1 (28NC1). Divergence is shown using appropriate single-letter designations. Gaps made for the most informative comparison of amino acid sequences are indicated by a dash (-). Putative transcription promoter sites (-10 and -35) and ribosome binding sites (RBS) are circled.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the cloning, sequencing and expression of homologous genes encoding an Ehrlichia canis protein with a molecular weight of 30 kilodaltons (kDa). A comparative molecular analysis of homologous genes was also performed in seven isolates of A.canis and the multigene family omp-1 E.chaffeensis. Two new genes encoding for 28 kDa proteins were identified - ECa28-1 and ECa28SA3. EC-28-1 has an 834-nucleotide open reading frame encoding a 278 amino acid protein (SEQ ID NO: 2) with an estimated molecular weight of 30.5 kDa. An N-terminal signal sequence has been identified: this confirms that the protein is characterized by post-translational modification into a mature protein weighing 27.7 kDa. ECa28SA3 has an 840 bp open frame that encodes a 280 amino acid protein (SEQ ID NO: 6).

Using PCR to amplify the 28 kDa protein genes of E.canis, the previously non-sequenced ECa28SA2 region was completely determined. Sequence analysis of ECa28SA2 revealed the presence of an 849-nucleotide open reading frame encoding a 283 amino acid protein (SEQ ID NO: 4). As a result of PCR amplification using primers specific for intergenic non-coding regions of 28 kDa protein genes, two previously separated loci were connected with the identification of a single locus (5592 bp), including all five protein genes weighing 28 kDa. Five proteins with a mass of 28 kDa, as was predicted, include signal segments causing the formation of mature proteins, and their amino acid homology level is 51-72%. Analysis of intergenic regions (spacers) indicated the putative promoter regions for each gene: this confirms that these genes can be expressed independently and differentially. The size of intergenic non-coding regions (28NCl-4) ranges from 299 to 355 bp. with a homology level of 48-71%.

The present invention relates to two new homologous 28 kDa protein genes in Ehrlichia canis - Esa28-1 and ECa28SA3 - and to the complete sequence of previously partially sequenced ECa28SA2. Also claimed is a multigenic locus encoding all five homologous external membrane proteins weighing 28 kDa Ehrlichia canis.

In one embodiment, the present invention provides a DNA sequence encoding an Ehrlichia canis 30 kDa immunoreactive protein. Preferably, the protein is characterized by an amino acid sequence selected from the group that includes SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6, and the gene is characterized by a nucleotide sequence selected from the group which includes SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, and is part of a polymorphic multigenic family. More preferably, the protein has an N-terminal signal sequence that is cleaved during post-translational processing, resulting in the formation of a mature protein with a mass of 28 kDa. Even more preferable, DNAs encoding proteins of 28 kDa are part of a single multigenic locus, the size of which is 5592 bp. and which encodes all five homologous proteins with a mass of 28 kDa in Ehrlichia canis.

In another embodiment, the present invention relates to an expression vector comprising a gene that encodes an Ehrlichia canis 28 kDa immunoreactive protein and is capable of expressing a gene after the vector has been introduced into the cell.

In yet another embodiment, the present invention relates to a recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Preferably, the amino acid sequence is encoded by a nucleic acid sequence selected from the group which includes SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. Preferably, the recombinant protein comprises four variable segments that are located on the cell surface and are hydrophilic and antigenic. Even more preferably, the recombinant protein is an antigen.

In yet another embodiment, the present invention relates to a method for generating a recombinant protein, comprising the steps of forming a vector, which includes an expression section comprising a sequence that encodes an amino acid sequence selected from the group that includes SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 operably linked to the promoter; cell vector transfection; and culturing the cell under conditions effective for expression of the expression site.

Also, the present invention can be described in some variations as a method of suppressing an Ehrlichia canis infection in a subject, comprising the steps of: identifying a subject suspected of being affected or infected by Ehrlichia canis; and administering a composition containing an antigen of 28 kDa of Ehrlichia canis in an amount effective to suppress Ehrlichia canis infection. Suppression can occur through any pathway, for example, through stimulation of a subject's humoral or cellular immune responses, or through other pathways, such as suppressing normal 28 kDa antigen function or even competing with an antigen for interacting with some agent in the subject's body.

In accordance with the present invention can be applied standard methods of molecular biology, microbiology and recombinant DNA technologies known to specialists in this field of technology. Such methods are fully described in the scientific literature: see, for example, Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning: A Practical Approach", Volumes I & II (D.N. Glover, ed., 1985); "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Nucleic Acid Hybridization" [B.D. Hames & S.L. Higgins, eds. (1985)]; "Transcription and Translation" [B.D. Hames & S.L. Higgins, eds. (1984)]; "Animal Cell Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells and Enzymes" [IRL Press (1986)]; B. Perbal, "A Practical Guide to Molecular Cloning" (1984).

So, as they appear in this text, the following terms should be defined in accordance with the following.

“Replicon” means any genetic element (eg, plasmid, chromosome, virus) that functions as an autonomous unit of the in vivo DNA replication process: i.e. able to replicate under its own control.

“Vector” means a replicon, such as a plasmid, phage or cosmid, to which another DNA segment can be attached in such a way as to allow replication of the attached segment.

“DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine), either in single-stranded form or in the form of a double-stranded helix. This term refers only to the primary and secondary structure of a molecule and does not limit it to any particular quaternary form. Thus, this term includes double-stranded DNA, found, inter alia, in linear DNA molecules (for example, restriction fragments, viruses, plasmids and chromosomes). When discussing the structure in this text, in accordance with the accepted norm, only a sequence is given in the direction from 5 'to 3' in the form of a non-transcribed (i.e. coding) DNA strand (i.e. a strand having the same sequence as that of mRNA )

A “coding sequence" of DNA refers to a double-stranded DNA sequence that is transcribed and translated into an in vivo polypeptide when it comes to the control of suitable regulatory sequences. The boundaries of the coding sequence are determined by the start codon at the 5’ end (N-end) and the stop codon at the 3’ end (C-end). A coding sequence may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNAs, genomic DNA sequences of eukaryotes (e.g., mammals), and even synthetic DNA sequences. The polyadenylation signal and transcription termination site should usually be located on the 3’ side of the coding sequence.

Transcriptional and translational control sequences are regulatory DNA sequences, such as promoters, enhancers, polyadenylation signals, terminators and the like, which allow expression of the coding sequence in the host cell.

“Promoter sequence” means a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of the coding sequence below (in the 3 ’direction). For the purposes of determination in the present invention, the promoter sequence is linked at its 3 ′ end to the transcription initiation site and continues upward (in the 5 ’direction) so as to include the minimum number of nucleotides or elements necessary for transcription initiation at levels clearly exceeding the background level. Within the promoter sequence, one can find the site of transcription initiation, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Often, but not always, promoters in eukaryotes include TATA boxes and CAT boxes. Prokarrhythmic promoters include Shine-Dalgarno sequences in addition to the consensus sequences at positions -10 and -35.

An “expression control sequence” means a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. The coding sequence is “under control” of the transcriptional and translational regulatory sequences in the cell when the RNA polymer transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

A “signal sequence” may be included next to the coding sequence. This sequence encodes a signal segment located in the N-terminal portion of the polypeptide and provides the host cell with the transfer of the polypeptide to the cell surface or secretion of the polypeptide into the environment, and this signal segment is cleaved by the host cell before the protein leaves the cell. Signal sequences can be detected in association with various proteins native to prokaryotes and eukaryotes.

The term “oligonucleotide”, as used herein, denotes the probe of the present invention, defines a molecule consisting of two or more ribonucleotides, preferably more than three. Its exact length will depend on many factors, which, in turn, depend on the final function and use of the oligonucleotide.

The term “primer” as used in this text means an oligonucleotide, both naturally occurring in the form of a purified restriction fragment, and synthetically obtained, which can act as a site for the initiation of synthesis when it comes to conditions in which the synthesis of a complementary product from the primer starts nucleic acid chains (template), i.e. in the presence of nucleotides and an inducing agent such as DNA polymerase, and at suitable temperature and pH. The primer can be either single-stranded or double-stranded and must be long enough to seed the synthesis of the desired finished product in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer and the method used. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide primer typically consists of 15-25 or more nucleotides, although it may include fewer nucleotides.

The primers in this application are selected as “substantially” complementary to the different chains of a particular DNA target sequence. This means that the primers must be complementary enough to hybridize with their respective chains. Therefore, the primer sequence does not have to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be attached to the 5'-end of the primer, and the rest of the primer sequence will be complementary to this chain. Alternatively, non-complementary nucleotides or longer sequences can be dispersed along the primer, if only this primer sequence is essentially complementary to the sequence or hybridizes with it, thereby forming a matrix for the synthesis of the completed product.

A cell is then considered to be “transformed” exogenous or heterologous DNA when such DNA has been introduced into the cell. Transforming DNA may integrate (by covalent binding) or may not integrate into the cell genome. In prokaryotes, yeasts, and mammalian cells, for example, transforming DNA can be maintained as an episomal element, such as a plasmid. In the case of eukaryotic cells, a stably transformed cell is a cell in which the transforming DNA becomes integrated into the chromosome so that it is transmitted to daughter cells as a result of chromosomal replication. Such stability is reflected in the ability of a eukaryotic cell to form cell lines or clones, including a population of daughter cells carrying transforming DNA. A “clone” is a population of cells formed from a single cell or ancestor as a result of mitosis. A “cell line” is a clone of a primary cell that is capable of stably growing in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75% (preferably at least about 80% and most preferably at least about 90% or 95%) of the nucleotides coincide in a particular region of the DNA sequences. Sequences that are essentially homologous can be identified by comparing the sequences using standard computer programs on the material of sequences from databases or using Southern blotting, for example, under the stringent hybridization conditions defined for this particular system. The determination of suitable hybridization conditions is known to those skilled in the art: see, for example, Maniatis et al., Cit. higher; "DNA Cloning", Vol. I & II, cit. higher; "Nucleic Acid Hybridization", cit. higher.

A “heterologous” region in a DNA construct is an identifiable segment of DNA in a larger DNA molecule for which there is no known association with this larger molecule in nature. Thus, when a heterologous region encodes a mammalian gene, the gene is usually flanked by DNA, which does not flank the DNA of the mammalian genome in the genome of the source organism. In another example, the coding sequence is a construct in which the coding sequence itself is not found in nature (e.g., cDNA in which the genomic coding sequence includes introns, or synthetic sequences including codons that are different from those in the native gene). Allelic variants or natural mutational events in connection with this definition do not form a heterologous DNA segment.

The labels most commonly used for these studies are radioactive elements, enzymes, chemical agents that fluoresce when irradiated with ultraviolet light, and others. A number of fluorescent materials are known that can be used as labels. They are, for example, fluorescein, rhodamine, auramine, Texas red, AMCA blue and lucifer yellow. A specific detection material is an anti-rabbit antibody generated in goats and coupled to fluorescein via isothiocyanate.

Proteins can also be labeled with a radioactive isotope or enzyme. A radioactive label can be detected using any of the currently available procedures. A preferred isotope can be selected from ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re.

Enzymatic labels are also applicable: they can be detected using any of the existing colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric methods. The enzyme is combined with the selected particle by reaction with an intermediate molecule such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes that can be used in these procedures are known and applicable. Preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase glucose oxidase and alkaline phosphatase. As an example, reference can be made to US Pat. Nos. 3654090, 3850752 and 4016043 in connection with the alternative materials and labeling methods set forth therein.

As used in this text, the term “host” is intended to include not only prokaryotes, but also eukaryotes, such as yeast, plant and animal cells. A recombinant DNA molecule or gene that encodes a 28 kDa Ehrlichia canis immunoreactive protein of the present invention can be used to transform a host using any of the methods well known to those skilled in the art. Most preferred is the use of a vector comprising the coding sequences of a gene encoding an Ehrlichia canis 28 kDa immunoreactive protein of the present invention for the purpose of transforming prokaryotes.

Prokaryotic hosts may be E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts are yeast, such as Pichia pastoris, mammalian cells and insect cells.

In general, expression vectors including promoter sequences that provide efficient transcription of the inserted DNA fragment are used in concert with the host. Typically, an expression vector includes a replication origin site, a promoter (s), a terminator (s), and specific genes that are capable of providing phenotypic selection in transformed cells. Transformed hosts can be fermented and cultured in accordance with methods known in the art in connection with achieving optimal cell growth.

The present invention includes substantially pure DNA encoding an Ehrlichia canis 28 kDa immunoreactive protein, the chain of which will hybridize at high stringency with a probe comprising a sequence of at least 15 consecutive nucleotides from SEQ ID NO: 1 or SEQ ID NO: 3, or SEQ ID NO: 5. The protein encoded by the DNA of the present invention may have at least 80% level of sequence identity (preferably 85%, more preferably 90% and most preferably 95%) with the amino acids listed SEQ ID NO: 2 or SEQ ID NO: 4, or SEQ ID NO: 6. More preferably, the DNA includes the coding sequence of nucleotides SEQ ID NO: 1 or SEQ ID NO: 3, or SEQ ID NO: 5, or a degenerate variant of such a sequence .

The probe with which the DNA of the present invention hybridizes preferably consists of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides and most preferably 100 nucleotides or more (up to full size) from the coding sequence of the nucleotides listed in SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 5, or their complements. Such a probe is useful for detecting expression of a 28 kDa Ehrlichia canis immunoreactive protein in a human cell using a method comprising the steps of: (a) contacting the mRNA obtained from the cell with a labeled hybridization probe; and (b) detecting hybridization of the probe with mRNA.

The present invention also relates to substantially pure DNA comprising a sequence of at least 15 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50 and most preferably all) from the nucleotide region listed in SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 5.

“High stringency” refers to DNA hybridization and washing conditions characterized by high temperature and low salt concentration, for example, washing conditions at 65 ° C. at a salt concentration of about 0.1 × SSC or their functional equivalents. For example, high stringency conditions may include hybridization at about 42 ° C in the presence of about 50% formamide; a first wash at about 65 ° C. in about 2 × SSC containing 1% SDS; followed by a second wash at about 65 ° C. in about 0.1 × SSC.

By “substantially pure DNA” is meant DNA that has ceased to be part of the environment in which DNA is found under native conditions, as a result of separation (partial or complete purification) from some or all of the molecules of this medium, or as a result of a change in sequences that flank the claimed DNA . Therefore, the term encompasses, for example, recombinant DNA that is inserted into a vector, into autonomously replicating plasmids or viruses, or into the genomic DNA of a prokaryotic or eukaryotic organism; or which exists as a single molecule (for example, cDNA or a fragment of a genomic or cDNA obtained by polymerase chain reaction (PCR) or by digestion with restriction endonucleases) independently of other sequences. It also encompasses recombinant DNA, which is part of a chimeric gene encoding an additional peptide sequence, such as a chimeric protein. Also covered is recombinant DNA, which includes part of the nucleotides listed in SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 5, which encodes an alternative splicing variant of a gene encoding an immunoreactive protein weighing 28 kDa Ehrlichia canis.

DNA can be characterized by at least about 70% sequence identity compared to the coding sequence of the nucleotides listed in SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 5, preferably at least 75% ( for example, at least 80%), and most preferably at least 90%. The identity of two sequences is a direct function of the number of matching or identical positions. When the position of a monomer in both sequences is occupied by the same monomer, for example, if this position is occupied by adenine in two DNA molecules, then they are identical in this position. For example, if 7 positions in a sequence of 10 nucleotides are identical to the corresponding positions in the second 10-nucleotide sequence, then these two sequences are characterized by 70% sequence identity. The length of the compared sequences as a whole should be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides and most preferably 100 nucleotides. Sequence identity is typically determined using a sequence analysis computer program (e.g., a sequence analysis software package from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University av., Madison, WI 53705).

The present invention relates to a vector comprising a DNA coding sequence for a gene that encodes a 28 kDa Ehrlichia canis immunoreactive protein, wherein said vector is capable of replicating in a host, which includes in functional communication: (a) a replication start site; (b) a promoter; and (c) a DNA sequence encoding said protein. Preferably, the vector of the present invention includes a portion of the DNA sequence shown in SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 5.

A “vector” can be defined as a replicating nucleic acid construct, for example a plasmid or viral nucleic acid. The vectors can be used to amplify and / or express a nucleic acid encoding an Ehrlichia canis 28 kDa immunoreactive protein. An expression vector is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of providing expression of the polypeptide in a cell. The need for such regulatory sequences will vary depending on the selected cell and the selected transformation method. In general, regulatory sequences include a transcription promoter and / or enhancer, suitable mRNA binding sites on ribosomes, and sequences that control transcription and translation termination. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising suitable transcriptional and translational regulatory elements: see, for example, the methods described by Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual "(2nd ed.). Cold Spring Harbor Press, NY. A gene and its transcriptional regulatory sequences are defined as “functionally linked” when the transcriptional regulatory sequences effectively control gene transcription. The vectors of the present invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the present invention are those derived from retroviruses, adenoviruses, adeno-associated virus, SV40 virus or herpes viruses.

By “substantially pure protein” is meant a protein that has been separated from at least some of the components that accompany it under native conditions. Typically, a protein is substantially pure if it is at least 60% by weight free of proteins and other naturally occurring organic molecules with which it is natively bound in vivo. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight. A substantially pure 28 kDa immunoreactive protein of Ehrlichia canis can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding an immunoreactive protein of 28 kDa Ehrlichia canis; or by chemical synthesis of protein. Purity can be determined using any suitable method, for example, column chromatography, such as immunoaffinity chromatography using an antibody specific for 28 kDa Ehrlichia canis immunoreactive protein, polyacrylamide gel electrophoresis, or HPLC analysis. A protein is substantially free of the components associated with it under native conditions, when it is separated from at least part of those polluting components that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system that is different from the cell from which it originates in vivo will, by definition, be substantially free of components associated with it in vivo. Consequently, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, or any other organism in which they are not found under natural conditions.

In addition to the substantially full-sized proteins, the present invention also provides fragments (e.g., fragments with antigenic properties) of an Ehrlichia canis 28 kDa immunoreactive protein (SEQ ID NO: 2 or SEQ ID NO: 4, or SEQ ID NO: 6). As used in this text, a “fragment” in an appendix to a polypeptide will denote at least 10 residues, more typically at least 20 residues, and more preferably at least 30 (eg 50) residues in length, but less than the complete, intact sequence. Fragments of a 28 kDa Ehrlichia canis immunoreactive protein can be obtained using methods known to those skilled in the art, for example, by catalytic cleavage of a native or recombinant 28 kDa Ehrlichia canis immunoreactive protein using recombinant DNA technologies using an expression vector that encodes a specific fragment of an immunoreactive protein weighing 28 kDa Ehrlichia canis, or by chemical synthesis. The ability of the candidate fragment to exhibit the property of a 28 kDa Ehrlichia canis immunoreactive protein (for example, the binding properties of an antibody specific for a 28 kDa Ehrlichia canis immunoreactive protein) can be evaluated using the methods described herein. Purified 28 kDa Ehrlichia canis immunoreactive protein or antigenic fragments of a 28 kDa Ehrlichia canis immunoreactive protein can be used to produce new antibodies or to analyze existing antibodies (for example, as positive controls in a diagnostic test) according to standard formulas known to those skilled in the art technicians. The present invention encompasses polyclonal antisera obtained using a 28 kDa Ehrlichia canis immunoreactive protein or a 28 kDa Ehrlichia canis immunoreactive protein fragment as an immunogen against, for example, rabbits. Use standard protocols for producing monoclonal and polyclonal antibodies, known to specialists in this field of technology. Monoclonal antibodies obtained using this procedure can be tested for their ability to identify recombinant Ehrlichia canis cDNA clones and distinguish them from known cDNA clones.

Further, the present invention relates to fragments of an immunoreactive protein weighing 28 kDa Ehrlichia canis, which are encoded at least in sections of SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 5, for example, products of alternative mRNA splicing or alternative processing events proteins or products in which a portion of the sequence has been deleted. A fragment or intact immunoreactive protein weighing 28 kDa Ehrlichia canis can be covalently linked to another polypeptide, which, for example, acts as a label, ligand, or antigenicity enhancer.

The term “pharmaceutically acceptable” means molecular components and compositions that do not cause an allergic or similar adverse reaction when administered to humans. A preparation of an aqueous composition that contains a protein as an active ingredient is well known in the art. Typically, such compositions are prepared in the form of injectable liquid solutions or suspensions: solid forms suitable also for reconstitution into a solution or suspension prior to injection can also be prepared. Also, the drug can be emulsified.

Protein may be included in the composition neutral or in salt form. Pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of proteins) that form inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic acids and the like. Salts formed by free carboxyl groups can also be obtained with inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or iron, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

After preparing the composition, the solutions can be administered in a manner appropriate to the dosage form, and in an amount that is therapeutically effective. Drugs can easily be administered in unit dosage forms, such as injection solutions.

For parenteral administration of an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent is made isotonic with a sufficient amount of salt or glucose. Such particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are well known to those skilled in the art in light of the disclosure of the present invention. For example, a single dose can be dissolved in 1 ml of an isotonic NaCl solution and either added to 1000 ml of hypodermolytic fluid or injected at the intended infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pp.1035-1038, pp .1570-1580). Some dose changes will be necessary depending on the condition of the subject being treated. In any case, the person responsible for the administration will be able to determine the appropriate dose for the individual patient.

As is well known in the art, a given polypeptide may vary in its immunogenicity. Often, therefore, it is necessary to combine an immunogen (e.g., a polypeptide of the present invention) with a carrier. An example and preferred carriers are slug hemocyanin (KLH) and human albumin. Other carriers may be various lymphokines and adjuvants, such as IL2, IL4, IL8 and others.

Means for coupling the polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. It should also be understood that the peptide can be coupled to the protein by genetic engineering methods that are well known in the art.

It is also well known in the art that the immunogenicity of a particular immunogen can be enhanced by the use of non-specific stimulants of the immune response, called adjuvants. Examples and preferred adjuvants are complete BCG, Detox (RIBI Immunochem Research Inc.), ISCOMS and aluminum hydroxide (Superphos, Biosector).

As used in this text, the term “complement” refers to a nucleic acid chain that will hybridize with the first nucleic acid sequence to form a double-stranded molecule under stringent conditions. Severe conditions are those conditions that provide hybridization between sequences of two nucleic acids having a high degree of homology, but exclude hybridization of random sequences. For example, hybridization at low temperature and / or high ionic strength is defined as low stiffness, and hybridization at high temperature and / or low ionic strength is defined as high stiffness. It is understood that temperature and ionic strength for the desired level of stiffness will be applied based on the specific probe sizes, sizes and nucleotide composition of the sequences, and the presence of formamide in the hybridization mixture.

As used herein, the term “artificially transformed” or “recombinant” cell is intended to mean a cell into which a recombinant gene has been introduced, such as a gene encoding the Ehrlichia chaffeensis antigen. Therefore, artificially transformed cells are different from native cells that do not include the introduced recombinant gene. Artificially transformed cells are thus cells carrying a gene or genes introduced by “human hands”. The introduced recombinant genes will be in the form of either a cDNA gene, or copies of the genomic gene, or will include genes located next to the promoter, which in vivo is not associated with a specific introduced gene. In addition, the recombinant gene can be integrated into the host genome or can be in the vector or in the bacterial genome by which the host cell has been transfected.

The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any way.

Example 1

Ehrlichi and purification

Ehrlichia canis (Florida strain and isolates Demon, DJ, Jake and Fuzzy) were provided by Dr. Edward Breitschwerdt (College of Veterinary Medicine, North Carolina Sate University, Raleigh, NC). E.canis (Louisiana strain) was provided by Dr. Richard E. Corstvet (School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA) and E.canis (Oklahoma strain) was provided by Dr. Jacqueline Dawson (Centers of Disease Control & Prevention, Atlanta, GA). Reproduction of ehrlichia was performed in DH82 cells in DMEM culture medium supplemented with 10% fetal calf serum and 2 mM L-glutamine at 37 ° C. Intracellular growth in DH82 cells was monitored by the presence of E.canis morulae, for which cytological staining methods were used. Cells were harvested when 100% of the cells were infected with ehrlichia, and then pelletized in a centrifuge at 17,000 g for 20 minutes. Cell clots were destroyed in a Braun-Sonic 2000 sonicator twice at 40 W for 30 s on ice. Ehrlichy was purified as previously described (Weiss et al., 1975). The lysate was loaded into a discontinuous renographin gradient of 42% -36% -30% and centrifuged at 80,000 g for 1 hour. The heavy and light bands containing ehrlichia were collected and washed with sucrose-phosphate-glutamate buffer (SFH, 218 mm sucrose, 3.8 mm KH ₂ PO ₄ , 7.2 mm K ₂ NRA ₄ , 4.9 mm glutamate, pH 7 , 0) and pelletized by centrifugation.

Example 2

Nucleic acid preparation

The genomic DNA of Ehrlichia canis was prepared by resuspending purified with renografin ehrlichia in 600 μl of 10 mm Tris-Hcl buffer (pH 7.5) with 1% sodium dodecyl sulfate (SDS, w / v) and 100 ng / ml proteinase-K in accordance with previously described (McBride et al., 1996). This mixture was incubated for 1 hour at 56 ° C, and the nucleic acids were extracted twice with a mixture of phenol / chloroform / isoamyl alcohol (24: 24: 1). DNA was pelletized by absolute ethanol precipitation, washed once with 70% ethanol, dried and resuspended in 10 mM Tris (pH 7.5). Plasmid DNA was purified using the High Pure Plasmid Isolation Kit (Boehringer Mannheim, Indianapolis, IN), and PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Santa Clarita, CA).

Example 3

PCR amplification of 28 kDa E.canis protein genes

Plots of the E.canis ECa28-1 gene selected for PCR amplification were selected based on the homology revealed (> 90%) in the consensus sequence obtained by matching the sequences of the p28 genes E. chaffeensis and map-1 Cowdria in the Jotun-Hein program ruminantium. Forward primer 793 (5'-GCAGGAGSTGTTGGTTASTS-3 ') (SEQ ID NO: 16) and reverse primer 1330 (5'-CCTTCCTCCAAGTTCTATGCC-3') (SEQ ID NO: 17) corresponded to nucleotides 313-332 and 823-843 MAP- 1 C. ruminantium and nucleotides 307-326 and 834-814 P28 of E. chaffeensis. E.canis DNA (North Carolina -Jake isolate) was amplified using primers 793 and 1330 with a thermopolymerase cycle dynamics of 2 minutes at 95 ° C and 30 cycles of 30 seconds at 95 ° C, 1 minute at 62 ° C, 2 minutes at 72 ° С followed by completion at 72 ° С for 10 minutes and storage at 4 ° С. PCR products were analyzed on a 1% agarose gel. This PCR amplified product was sequenced directly using primers 793 and 1330.

Primers specific for the ECa28SA2 gene — designed 46f (5’-ATATACTTCCTACCTAATGTCTCA-3 ’, SEQ ID NO: 18) and primer 1330 (SEQ ID NO: 17) —were used to amplify the targeted region. The amplified product was gel purified and cloned into a TA cloning vector (Invitrogen, Santa Clarita, CA). The clone was sequenced in two directions using primers: reverse M13 from vector, 46f, ECa28SA2 (5'-AGTGCAGAGTCTTCGGTTTC-3 ', SEQ ID NO: 19), ECa5.3 (5'-GTTACTTGCGGAGGACAT-3', SEQ ID NO: 20 ) DNA was amplified in a thermopolymerase cycle: 2 minutes at 95 ° C and 30 cycles of 30 seconds at 95 ° C, 1 minute at 48 ° C, 1 minute at 72 ° C, followed by completion at 72 ° C for 10 minutes and stored at 4 ° C.

Example 4

Sequencing of unknown 5’ and 3’ segments of the ECa28-1 gene

The full-length sequence of EC-28-1 was determined using the Universal Genome Walker Kit (Clontech, Palo Alto, CA) in accordance with the words provided by the manufacturer. E.canis genomic DNA (Jake isolate) was completely digested with five restriction enzymes (DraI, EcoRV, PvuII, ScaI, StuI), which form blunt ends in DNA. The adapter proposed by the kit (AP1) was ligated to each end of E.canis DNA. Genomic libraries were used as templates for detecting an unknown DNA sequence of the EC-28-1 gene using PCR using a primer complementary to a known part of the EC-28-1 sequence and a primer specific for the AP1 adapter. The primers specific for EC-28-1, used for “walking” along the genome, were constructed using the known DNA sequence obtained by PCR amplification of EC-28-1 with 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 394C (5'-TTACCTATGATCCTGTGGAAATGC-3'; nucleotides 710-687, SEQ ID NO: 22) were used in conjunction with the provided AP1 for amplification of unknown 5'- and 3'-segments of the EC-28-1 gene using PCR. The PCR product corresponding to the 5'-segment of the EC-28-1 gene, amplified with primers 394C and AP1 (2000 bp), was sequenced in the same direction with primer 793C (5'-GAGTAACCAACAGCTCCTGC-3 ', SEQ ID NO: 23) . The PCR product corresponding to the 3’-segment of the EC-28-1 gene amplified with primers 394 and AP1 (580 bp) was sequenced in two directions in the same primers. Sequences of non-coding regions of the 5'- and 3'-segments adjacent to the open coding frame were sequenced, and the primers EC28OM-F (5'-TCTACTTTGCACTTCCACTATTGT-3 ', SEQ ID NO: 24) and EC28OM-R (5'-ATTSTTTTTGSSASTATTTTTTTTTTTTTTTTTTTTT , SEQ ID NO: 25), complementary to these segments, were designed to amplify the full-length EC-28-1 gene.

Example 5

Sequencing of E.canis Isolates

DNA was sequenced using an ABI Prism 377 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Full-length ECa28-1 genes from seven E.canis isolates (four from North Carolina and one each from Oklahoma, Florida, and Louisiana) were amplified by PCR with EC28 OM-F primers (SEQ ID NO: 24) and EC28 OM-R (SEQ ID NO: 25) in the thermopolymerase cycle: 5 minutes at 95 ° C and 30 cycles of 30 seconds at 95 ° C, 1 minute at 62 ° C and 2 minutes at 72 ° C with completion at 72 ° C for 10 minutes. The resulting PCR products were sequenced in both directions using the same primers.

Example 6

Cloning and expression of EC-28-1 E.canis

The full-length E.canis ECa28-1 gene was PCR amplified with EC28OM-F and EC28OM-R primers and cloned into the pCR2.1-TOPO TA cloning vector to obtain the desired group of restriction enzyme digestion sites (Invitrogen, Carlsbad, CA). The insert was excised from pCR2.1-TOPO by BstXl restriction enzyme and ligated into pcDNA3.1, a eukaryotic expression vector (Invitrogen, Carlsbad, CA) designated pcDNA3.1 / EC28, for further study. Plasmid pcDNA3.1 / EC28 was amplified and the gene was excised by treatment with two KpnI / XbaI restriction enzymes and directly ligated into the prokaryotic expression vector pThioHis (Invitrogen, Carlsbad, CA). The clone (designated pThioHis / EC28) produced the recombinant chimeric thioredoxin protein in Escherichia coli BL21. Recombinant chimeric protein was subjected to coarse purification in the insoluble phase by centrifugation. A control chimeric thioredoxin protein was purified from soluble cell lysates under native conditions using Ni-NTA rotating columns (Qiagen, Santa Clarita, CA).

Example 7

Western immunoblot analysis

Recombinant E.canis ECa28-1 chimeric protein was subjected to SDS-polyacrylamide gel (SDS-PAGE) electrophoresis in gradient gels 4-15% with Tris-Hcl (Bio-Rad, Hercules, CA) and transferred onto pure nitrocellulose (Schleicher & Schuell , Keene, NH) using a semi-dry cell transfer method (Bio-Rad, Hercules, CA). The membrane was incubated with antiserum from a recovering dog infected with E.canis, diluted 1: 5000, for 1 hour, washed and then incubated with anti-dog IgG (H&L) secondary antibody, purified by an affinity method coupled with alkaline phosphatase, diluted 1: 1000 for 1 hour (Kirkegaard & Perry Laboratories, Gaithersburg, MD). The bound antibody was visualized using 5-bromo-4-chloro-3-indolylphosphate / blue-blue tetrazolium (BCIP / NBT) as substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Example 8

Southern blot analysis

To determine whether multiple genes homologous to EC-28-1 are present in the E.canis genome, Southern blot genomic analysis was performed using a standard procedure (Sambrook et al., 1989). The E.canis genomic DNA was completely digested with each of the restriction enzymes BanII, EcoRV, HaeII, KpnI and SpeI, which do not have sites within the EC-28-1 gene, as well as with the restriction enzyme AseI, which cuts the EC-28-1 gene into nucleotides 34, 43 and 656. The probe was obtained by PCR amplification with primers EC28OM-F and EC28OM-R and labeled with digoxigenin (DIG) deoxyribonucleotide triphosphates (dNTP) (Boehringer Mannheim, Indianapolis, IN) and digested with restriction enzyme AseI. The digested probe (566 bp) was separated by agarose gel electrophoresis, gel purified and then used for hybridization. The fully digested E.canis genomic DNA was electrophoresed, transferred to a nylon membrane (Boehringer Mannheim, Indianapolis, IN) and hybridized at 40 ° C for 16 hours with a DIG-labeled probe of the EC-28-1 gene in DIG Easy Hyb buffer in accordance with the words manufacturer (Boehringer Mannheim, Indianapolis, IN). A bound probe was detected with an anti-digoxigenin antibody coupled to alkaline phosphatase and a luminescent substrate (Boehringer Mannheim, Indianapolis, IN) and exposed to a special BioMax film (Eastman Kodak, Rochester, NY).

Example 9

Sequence Analysis and Comparison

The p28 DNA sequences of E. chaffeensis and C. ruminantium map-1 were obtained from the National Center Biotechnology Information (NCBI) (Internet site http://www.ncbi.nlm.nih.gov/Entrez). Nucleotide and decrypted amino acid sequences, as well as protein and phylogenetic analysis were performed using the LASERGENE program (DNASTAR Inc., Madison, WI). Post-translational processing analysis was performed using the McGeoch & von Heijne method of searching for a signal sequence using the PSORT program (McGeoch, 1985; von Heijne, 1986) (Internet site: PRIVATE HREF - http://www.imcb.osaka-u.ac. jp / nakai / form.htm; MACROBUTTON HtmlResAnchor http://www.imcb.osaka-u.ac.jp/nakai/form. htm).

GenBabk's deposit numbers for the nucleotide and amino acid sequences of the E.canis ECa28-1 genes described in this study are as follows: Jake - AF082744; Louisiana - AF082745; Oklahoma - AF082746; Demon - AF082747; DJ - AF082748; Fuzzy - AF082749; Florida - AF082750.

Sequence analysis of EC-28-1 from seven different E.canis strains was performed with primers designed to amplify the complete gene. The analysis showed that the sequence of this gene was conserved among isolates originating from North Carolina (four), Louisiana, Florida, and Oklahoma.

Example 10

PCR amplification, cloning, sequencing and expression of EC-28-1

Comparison of the nucleotide sequences of p28 E. chaffeensis and map-1 Cowdria ruminantium using the Jotun-Hein program allowed us to obtain a consensus sequence with segments of a high level of homology (> 90%). These homologous segments (nucleotides 313-332 and 823-843 map-1 of C. ruminantium; nucleotides 307-326 and 814-834 p28 of E. chaffeensis) were selected as sites for primer annealing in PCR amplification. PCR amplification of E.canis EC-28-1 and E. chaffeensis p28 gene was performed using primers 793 and 1330 to obtain a 518 bp PCR product. The nucleotide sequence of the E.canis PCR product was determined by direct sequencing of the product with primers 793 and 1330. The sequence analysis revealed an open reading frame encoding a protein of 170 amino acids, and the 518 bp sequence obtained by PCR amplification E.canis, with the p28 DNA sequence of E. chaffeensis indicated a similarity level of more than 70%: this indicates the homology of these genes. PCR with an adapter with primers 394 and 793C was carried out in order to determine the 5'- and 3'-segments of the sequence of the full-sized gene. Primer 394 gave four PCR products (3 kb, 2 kb, 1 kb, and 0.8 kb): a product of 0.8 t in length. bp sequenced in two directions using primers 394 and AP1. The decrypted sequence overlapped with the 3’-end of 518-bp. product, extending the open reading frame by 12 bp towards the stop codon. An additional 625 bp was also sequenced. non-coding sequence at the 3’ end of the EC-28-1 gene. Primer 394C was used to amplify the 5 ′ end of the EC-28-1 gene along with the primer AP1 provided. Amplification with these primers resulted in three PCR products (3.3 kb, 3 kb and 2 kb). A fragment of 2 kb sequenced in one direction by primer 793C. The sequence yielded the putative start codon of the EC-28-1 gene and a complete open reading frame of 834 bp encoding a protein of 278 amino acids. An additional 144 bp was received. the read sequence in the 5’-untranslated segment of the EC-28-1 gene. The EC28 OM-F and EC28 OM-R primers were designed according to complementary non-coding segments flanking the EC-28-1 gene.

The PCR product amplified with these primers was sequenced directly with the same primers. The complete DNA sequence (SEQ ID NO: 1) for the E.canis EC-28-1 gene is shown in FIG. The ECA28-1 PCR fragment amplified with these primers included a full-size open reading frame and 17 additional amino acids along the 5’ non-coding primer segment. The gene was directly subcloned into the pThioHis expression vector, and E. coli cells (BL21) were transformed with this construct. The expressed chimeric protein EC-28-1-thioredoxin was found to be insoluble. The expressed protein included an additional 114 amino acids associated with thioredoxin, 5 amino acids of the enterokinase recognition site, and 32 amino acids of the multiple cloning site and a 5 ′ non-coding primer segment at the N-terminus. The antiserum of a recovering dog infected with E.canis recognized the expressed recombinant protein but did not interact with the control thioredoxin (Figure 2).

Example 11

Sequence homology

ECA28-1 nucleic acid sequences (834 bp) and E. chaffeensis omp-1 family genes, including signal sequences (EC-28-1, omp-1A, B, C, D, E and F), were compared using the method Clustal for the purpose of analyzing homology between these genes (the comparison itself is not shown). The nucleic acid homology was equally conservative (68.9%) between EC-28-1 and p28 of E. chaffeensis, and omp-1F. Genes of other putative external membrane proteins from the omp-1 family of E. chaffeensis, omp-1D (68.2%), omp-1E (66.7%), omp-1C (64.1%), map-1 Cowdria ruminantium ( 61.8%), the E.canis protein-1 gene with a mass of 28 kDa (60%) and the 28 kDa protein-2 gene (partially) (59.5%) were also homologous to EC-28-1. The smallest homology of nucleotide sequences (45.1%) was observed between omp-1B of E. chaffeensis and EC-28-1.

A comparison of the predicted amino acid sequences of EC-28-1 (SEQ ID NO: 2) and P28 of E. chaffeensis revealed amino acid substitutions leading to the appearance of four variable segments (VR). Replacements or deletions in the amino acid sequence and the location of the variable segments in EC-28-1 and in members of the OMP-1 family of E. chaffeensis were identified (Fig. 3). A comparison of amino acids, including the signal segment, showed that EC-28-1 shows the highest level of homology with OMP-1F (68%) from the OMP-1 family of E.chaffeensis, then with P28 E.chaffeensis (65.5%), OMP-1E (65.1%), OMP-1D (62.9%), OMP-1C (62.9%), MAP-1 Cowdria ruminantium (59.4%), protein-1 with a mass of 28 kDa E.canis (55 , 6%) and protein-2 weighing 28 kDa (partial) (53.6%), and OMR-1B (43.2%). Phylogenetic reconstructions based on amino acid sequences show that EC-28-1 and C. ruminantium MAP-1, E. chaffeensis OMP-1 proteins, and 28 kD E.canis proteins 1 and 2 (partial) are related (Fig. 4).

Example 12

Predicted Surface Probability and Immunoreactivity

Analysis of E.canis EC-28-1 by the profiles of hydropathy and hydrophilicity made it possible to predict the sites located on the surface of the EC-28-1 molecule (Fig.6). Eight major surface-located regions composed of 3–9 amino acids were identified in EC-28-1 and were similar to the surface region profile of E. chaffeensis P28 (FIG. 6). The five largest surface sites of EC-28-1 were located in the N-terminal part of the protein. Hydrophilic sites located on the surface were found in all four variable segments of the EC-28-1. Ten T cell motifs were predicted in EC-28-1 using the Rothbard-Taylor method (Rothbard & Taylor, 1988), and high antigenicity of EC-28-1 was predicted using the Jameson-Wolf antigenicity estimation algorithm (Fig. 6) (Jameson & Wolf, 1988). A similarity of antigenicity and T-cell motifs was found between EC-28-1 and P28 of E.chaffeensis.

Example 13

Identification of homologous genomic copies of the EC-28-1 gene

Genomic analysis by Southern blotting of E.canis DNA, completely and individually digested with restriction enzymes BanII, EcoRV, HaeII, KpnI, SpeI, for which there are no recognition sites within the EC-28-1 gene, as well as AseI restriction enzyme, for which there are restriction sites in nucleotides 34, 43 and 656, showed the presence of at least three homologous copies of the EC-28-1 gene (Figure 5). Although EC-28-1 includes an internal AseI restriction enzyme recognition site, the DIG-labeled probe used in the hybridization experiment labeled a region of the gene within a single DNA fragment resulting from AseI processing of this gene. The restriction enzyme digestion with AseI led to the formation of 3 bands (approximately 566 bp, 850 bp and 3 bp long) that hybridized with the EC-28-1 DNA probe, indicating the presence of multiple homologous genes EC-28-1, in the genome. The restriction enzyme digestion with EcoRV and SpeI yielded two bands that hybridized with the probe of the EC-28-1 gene.

Example 14

Identification of a 28 kDa protein gene locus

Specific primers designated ECaSA3-2 (5'-CTAGGATTAGGTTATAGTATAAGTT-3 ', SEQ ID NO: 26) corresponding to regions within ECa28SA3, and primer 793C (SEQ ID NO: 23), which binds to the region of ECa28-1, was used for amplification of the intergenic spacer between the SA3 and EC-28-1 genes. 800 bp product was sequenced using the same primers. DNA was amplified in a thermopolymerase scheme: 2 minutes at 95 ° C and 30 cycles of 30 seconds at 95 ° C, 1 minute at 50 ° C, 1 minute at 72 ° C, followed by completion at 72 ° C for 10 minutes and stored at 4 ° C.

Example 15

PCR amplification of 28 kDa protein genes and identification of a multigenic locus

In order to specifically amplify possible unknown genes located downstream from ECa28SA2, the amplification was performed using primer 46f and primer 1330, which corresponds to the conserved segment from the 3’ end of the ECa28-1 gene. According to these primers, a PCR product of 2 kb was amplified, in which there were two open reading frames. The first open reading frame included a known gene region — ECaSA2 — and a previously unsequenced 3 ′ gene region. Down from ECaSA2, an additional non-identical, but homologous, 28 kDa protein gene was found - it was designated ECa28SA3. Two known loci were amplified with an SA3-2 primer specific for the 3 ′ end of the ECa28SA3 gene, which was used in combination with the 793C reverse primer that binds to the 5 ′ end of the EC-28-1. The 800 bp PCR product was amplified, which included the 3'-end of ECa28SA3, the intergenic spacer between ECa28SA3 and EC-28-1 (28NC3), and the 5'-end of EC-28-1 connecting previously separate loci (Fig. 8). 849 bp open reading frame ECa28SA2 encodes a 283 amino acid protein, and ECa28SA3 has an 840 bp open reading frame encoding a 280 amino acid protein. The intergenic non-coding region between ECa28SA3 and ECa28-1 was 345 bp in length. (Fig.7 and 8).

Example 16

Nucleotide and amino acid homology

The nucleotide and amino acid sequences of all five protein genes weighing 28 kDa E.canis were compared using the Clustal method to analyze homology between genes. Homology of nucleic acids ranged from 58-75%, and the similar homology of amino acid sequences was 67-72% when comparing genes encoding 28 kDa proteins of E.canis (Fig. 9).

Example 17

Plots controlling transcription promoters

Intergenic regions between 28 kDa protein genes were analyzed for the presence of promoter sequences by comparison with the consensus promoter regions of Escherichia coli and the E. chaffeensis promoter (Yu et al., 1997; McClure, 1985).

Putative promoter sequences, including RBS, -10 and -35 regions, were identified in four intergenic sequences corresponding to the ECa28SA2, ECa28SA3, ECa28-1 and ECa28-2 genes (Figure 10). The non-coding region upstream of the ECa28SA1 gene is unknown and has not been investigated.

Example 18

N-terminal signal sequence

Analysis of amino acid sequences showed that full-length ECa28-1 E.canis has a decrypted molecular weight of 30.5 kDa, and full-sized ECa28SA3 has a decrypted molecular weight of 30.7 kDa. Both proteins include the predicted N-terminal signal segment of 23 amino acids (MNCKKILITTALMSLMYYAPSIS, SEQ ID NO: 27), which is similar to that predicted for P28 E.chaffeensis (MNYKKILITSALISLISSLPGVSFS, SEQ ID NO: 28) and proteins of the OMP-1 family (Yu et al al. ., 1998; Ohashi et al., 1998b). A preferred signal peptidase cleavage site (SIS; serine-X-serine) (Oliver, 1985) is found in amino acids 21, 22 and 23 of ECa28-1. There was also an additional site of putative cleavage at amino acid position 25 (MNCKKILITTALISLMYSIPSISSFS, SEQ ID NO: 29), identical to the predicted cleavage site in P28 of E. chaffeensis (SFS), which caused the formation of mature EC-28-1 protein with a predicted molecular weight of 27.7 kDa. The signal cleavage site of the previously published partial sequence of ECa28SA2 is assumed to be at the 30th amino acid. However, signal sequence analysis predicted that the EC-28SA1 had a non-cleavable signal sequence.

Summary

Proteins with similar molecular weights were identified and cloned in several rickettsia agents, including E.canis, E. chaffeensis, and C. ruminantium (Reddy et al., 1998; Jongejan et al., 1993; Ohashi et al., 1998). Previously, a single locus was described in Ehrlichia chaffeensis with 6 homologous p28 genes and two loci in E.canis, each of which includes several homologous protein genes weighing 28 kDa.

The present invention has shown the cloning, expression, and characterization of genes encoding a mature 28 kDa E.canis protein that are homologous to the multigene family ot-1 E. chaffeensis and the marm-1 gene C. ruminantium. Two new 28 kDa protein genes were identified - EC-28-1 and EC-28SА3. Another E.canis 28 kDa protein gene, SCa28SA2, which was partially sequenced previously (Reddy et al., 1998), was completely sequenced in the present invention. Also claimed is the identification and characterization of a single E.canis locus, including all five protein genes weighing 28 kDa E.canis.

A protein with a mass of 28 kDa E.canis is homologous to the OMP-1 family of E. chaffeensis and the protein MAP-1 of C. ruminantium. The most homologous proteins with a mass of 28 kDa E.canis (ECa28SA3, ECa28-1 and ECa28-2) are arranged in a tandem locus. The homology of these proteins ranged from 67.5% to 72.3%. The divergence level of these proteins weighing 28 kDa ranged from 27.3% to 38.6%. Proteins weighing 28 kDa E.canis ECa28SAl and ECa28SA2 were characterized by the least homology with a homology level in the range from 50.9% to 59.4% and with a divergence from 53.3% to 69.9%. The differences between the genes are confined mainly to four hypervariable regions: this confirms that these regions are located on the surface and are under a selection press with the participation of the immune system. The conservatism of ECa28-1 has been described in seven isolates of E.canis (McBride et al., 1999): this confirms that E.canis can be characterized by clonal reproduction in North America. On the contrary, a significant level of divergence of p28 proteins was reported in isolates of E.chaffeensis (Yu et al., 1998).

All proteins with a mass of 28 kDa in E.canis are thought to undergo post-translational processing, turning from a protein of 30 kDa into a mature protein of 28 kDa. Recently, the signal sequence was identified in P28 of E.chaffeensis (Yu et al., 1998), and sequencing of the N-terminal amino acids confirmed that this protein is subject to post-translational processing, which consists in cleaving the signal sequence to form a mature protein (Ohashi et al., 1998 ) The leader sequences of OMP-1F and OMP-1E are also thought to be leader signal peptides (Ohashi et al., 1998). The signal sequences identified in OMP-1F, OMP-1E, and P28 in E. chaffeensis are homologous to the leader sequence of a 28 kDa E.canis protein. The promoter sequences of the p28 genes have not been experimentally determined, but putative promoter regions have been identified by comparing the consensus sequences of the RBS, -10 and -35 promoter segments in E. coli and other Ehrlichia (Yu et al., 1997; McClure, 1985) . Such promoter sequences should provide the gene with potential transcription and translation: this confirms that these genes can be expressed differently in the host. The persistence of infection in dogs can be associated with differentiated expression of p28 genes, leading to in vivo antigenic transformations, which allows the pathogen to avoid the immune response.

Protein genes with a mass of 28 kDa E.canis were found to be characterized by homology of nucleotide and amino acid sequences with genes of the omp-1 family of E. chaffeensis and with the marm-1 gene of C. ruminantium. In previous studies, a protein of 30 kDa was identified in E.canis, which interacts with the antiserum to E. chaffeensis obtained from recovering dogs, but it is believed that they are not the same by antigenic properties (Rikihisa et al., 1994). A comparison of amino acid substitutions in four variable regions of 28 kDa E.canis proteins confirms this possibility. Along with these data, it is also confirmed that the amino acids responsible for the antigenic differences between P28 E.canis and E.chaffeensis are located in these variable regions and are readily available to the immune system. It was reported that immunoreactive peptides were located in variable segments of 28 kDa proteins of C. ruminantium, E. chaffeensis and E. canis (Ready et al., 1998). Analysis of P28 by E.canis and E.chaffeensis showed that all variable segments have predicted amino acids located on the surface of the molecule. Dog analysis showed no cross-reactivity between E.canis and E.chaffeensis (Dawson & Ewing, 1992). This observation may be related to the antigenic differentiation of variable segments in the composition of P28, as well as in other immunologically important antigens of these species of ehrlichia. Another study found that the human recovery stage antiserum in patients infected with E. chaffeensis recognizes 29/28 kDa E. chaffeensis protein (s) and also reacts with homologous E.canis proteins (Chen et al., 1997) . Homologous and cross-reactive protein epitopes of 28 kDa E.canis and P28 E. chaffeensis are presumably recognized by the immune system.

Proteins weighing 28 kDa E.canis can be important immunoreactive antigens. Some reports have shown that an antigen of 30 kDa E.canis exhibits intense immunoreactivity (Rikihisa et al., 1994; Rikihisa et al., 1992). Antibodies in the recovery stage antisera obtained from humans and domestic dogs are characterized by significant reactivity to E. chaffeensis and E. canis proteins in this size range: this confirms that they can be important immunoreactive antigens (Rikihisa et al., 1994; Chen et al., 1994; Chen et al., 1997). In addition, antibodies to proteins with a mass of 30, 24, and 21 kDa were formed in the early stages of the formation of an immune response to E.canis (Rikihisa et al., 1994; Rikihisa et al., 1992): this confirms that these proteins can be especially important for immune responses in the acute stage of the disease. Recently, a family of homologous genes encoding external membrane proteins with molecular weights of 28 kDa was identified in E. chaffeensis, and in mice immunized with recombinant P28 E. chaffeensis, immunity against homologous infection apparently developed (Ohashi et al., 1998). Protein P28 E.chaffeensis has been shown to be present on the outer membrane, and according to immunoelectron microscopy data, P28 was mapped onto the surface of this organism: therefore, it was confirmed that it can act as an adhesion protein (Ohashi et al., 1998). It is likely that the proteins with a mass of 28 kDa E.canis identified in this study have the same localization and possibly perform similar functions.

Comparison of EC-28-1 in different strains of E.canis showed that the gene is apparently completely conservative. Studies involving E. chaffeensis have provided immunological and molecular evidence for EC-28-1 divergence. Patients infected with E. chaffeensis showed varying immunoreactivity for 29/28 kDa proteins: this confirms their antigenic differentiation (Chen et al., 1997). Recently, molecular confirmation of the antigenic heterogeneity of the p28 gene was obtained in E. chaffeensis (Yu et al., 1998). Comparison of five E.chaffeensis isolates showed that two isolates (Sapulpa and St. Vincent) are 100% identical, but the other three isolates (Arkansas, Jax and 91HE17) diverged at least 13.4% at the level of amino acid sequences. The conservatism of EC-28-1 confirms that E.canis strains found in the USA can be genetically identical, i.e. 28 kDa protein E.canis is an attractive material for the development of vaccines against dog ehrlichiosis in the USA. Further analysis of E.canis isolates from outside the United States may provide information regarding the origin and evolution of E.canis. Conservatism of a protein of 28 kDa mass makes it an important potential candidate for effective serodiagnosis of dog ehrlichiosis.

The role of multiple homologous genes is currently unknown: however, the persistence of the E.canis pathogen in dogs can presumably be associated with antigenic variation due to the varying expression of 28 kDa homologous protein genes, thereby allowing E.canis to avoid immune system activity. The variability of the A. marginale msp-3 genes is partly responsible for the variability of the MSP-3 protein, which leads to the persistence of the pathogen (Alleman et al., 1997). Studies of the expression of 28 kDa E.canis protein genes in dogs with acute and chronic infection with this pathogen will provide information on the role of the 28 kDa protein gene family in pathogen persistence.

Any patents or publications mentioned in this application correspond to the level of specialists in the field of technology to which the present invention relates. These patents and publications are included here for information in the form of bibliographic references to the same extent that each individual publication is intended to be included for information in the form of a bibliographic reference.

For a person skilled in the art it will be obvious that the present invention can be easily applied to implement the objects, scope and advantages mentioned above, as well as those that are inherent in them. The present examples, along with the methods, procedures, treatments, molecules and specific compounds described herein, are representative of the preferred embodiments, are examples, and are not intended to limit the scope of the present invention in anything. Changes in it and other ways of using it will be clear to experts in the field of technology, which are covered by the essence of the present invention, defined by the scope of the claims.

The sequence listing is at the end of the description.

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Claims (12)

1. The DNA sequence encoding a protein of Ehrlichia canis with a molecular weight of 30 kilodaltons, where the specified protein is immunoreactive with serum against Ehrlichia canis and where the specified protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6. 2. The DNA sequence according to claim 1, where the specified protein has an N-terminal signal sequence shown in SEQ ID NO: 27. 3. The DNA sequence according to claim 2, where the specified protein is subjected to post-translational modification into a protein with a molecular weight of 28 kilodaltons. 4. The DNA sequence of claim 1, wherein said DNA has a sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 5. 5. The DNA sequence according to claim 1, where the specified DNA is contained in a single locus of Ehrlichia canis. 6. The DNA sequence according to claim 5, where the specified locus is a multigenic locus with a length of 5592 nucleotides. 7. The DNA sequence according to claim 6, where the specified locus encodes homologous proteins of Ehrlichia canis with a molecular weight of 28 kilodaltons. 8. The DNA sequence according to claim 7, where these homologous proteins of Ehrlichia canis with a molecular weight of 28 kilodaltons are selected from the group consisting of ECa28SA1, ECa28SA2, ECa28SA3, ECa28-1 and ECa28-2. 9. The vector for protein expression and containing the DNA sequence according to claim 1, where the specified vector is an expression vector capable of expressing a peptide or polypeptide encoded by a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, when the specified expression vector is introduced into the cell. 10. Recombinant protein for producing antibodies containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. 11. The recombinant protein of claim 10, where the specified amino acid sequence is encoded by a nucleic acid segment containing a sequence selected from SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. 12. The method of producing the recombinant protein of claim 10, comprising the following steps: obtaining a vector that contains an expression segment containing a sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6 operably linked to the promoter; transfection of a cell with a specified vector; culturing said cell under conditions providing expression of said expression segment.

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