JP4727059B2 - Utilization of nucleic acid molecule encoding Babesagibsoni P50 antigen and its expression product - Google Patents

Description

【００６１】
【発明の効果】
当該遺伝子はB.gibsoniの新規主要抗原タンパク質P50タンパク質をコードする遺伝子であり、当該タンパク質はイヌに対して抗原性を有することから、抗B.gibsoniワクチンとして使用し得る。また、本タンパク質もしくはそのフラグメントあるいは本タンパク質をコードする塩基配列をプローブとして用いることによって、従来近縁種との区別が困難であったB.gibsoni 原虫感染の診断がより特異的に実施し得る。
【００６２】
【配列表】

【図面の簡単な説明】
【図１】本発明の発現産物である組換えP50タンパク質を示す電気泳動図を示す。
レーン１：分子量マーカー、レーン２：精製組換えP50タンパク質、レーン３：精製gene 10タンパク質
【図２】組換えP50タンパク質免疫血清による天然型P50タンパク質の検出を示すイムノブロット像を示す。
レーン１：B.gibsoni感染赤血球、レーン２：正常犬赤血球
【図３】組換えP50タンパク質とB.gibsoni感染犬血清との反応性を示すイムノブロット像を示す。レーン１：精製組換えP50タンパク質、レーン２：精製gene 10タンパク質[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel protein derived from a protozoan of Babesia gibsoni (hereinafter abbreviated as B. gibsoni) and a gene encoding the protein.
[0002]
[Prior art]
B.gibsoni is an erythrocytic protozoan parasite mediated by ticks, and canine infections caused by this protozoan (B. gibsoni infection) are widely recognized in Asia, Africa, Europe and North America. In Japan, cases have been reported in almost all regions from Kyushu to Hokkaido, especially in western Japan.
[0003]
Dogs infected with B.gibsoni have symptoms such as loss of appetite, tendency to sleep, loss of energy, fever, anemia, vomiting, hematuria, hemoglobinuria, splenomegaly, hemoglobinemia, thrombocytopenia, systemic lymphadenopathy Indicates. Fever is caused by a pyrogen released during hemolysis or when the monocyte phagocytes respond to protozoan parasitic erythrocytes. The decrease in platelets is thought to be due to suppression of production in the bone marrow, platelet sequestration in the liver and spleen, increased platelet consumption, and immune-mediated platelet destruction. Dogs infected with B. gibsoni may also develop progressive anemia during surgery and immunosuppressive therapy, preventing recovery from the underlying disease and smooth treatment.
[0004]
Treatment of B. gibsoni infection is mainly carried out with diminazen preparations, but the use of this preparation is limited because it causes side effects such as neurological symptoms caused by cerebellar hemorrhage, liver damage, and kidney damage. ing.
[0005]
The diagnosis of this protozoan infection is currently performed mainly by confirming the protozoa with a blood smear, but this method has a problem of low detection sensitivity in chronically infected dogs. In addition, serological diagnostic methods using the fluorescent antibody method are also used, but cross-reaction with other erythrocyte parasites, Babesia canis, is observed, and its specificity is problematic.
[0006]
An effective vaccine for B. gibsoni infection has not yet been developed. The major factors include the difficulty of mass culture of B. gibsoni and the delay in searching for effective antigens related to infection protection. Against this background, the search for major antigens of B. gibsoni, the establishment of diagnostic methods with high sensitivity and specificity using recombinant proteins as antigens, and the development of effective recombinant vaccines are desired.
[0007]
[Problems to be solved by the invention]
The present invention provides a major antigen of B. gibsoni that can be used for the production of a vaccine for diagnosis and prevention of infection of B. gibsoni infection and a gene encoding the antigen.
[0008]
[Means for Solving the Problems]
The present inventors constructed a cDNA library of B. gibsoni, and identified and cloned a gene encoding a major antigen (hereinafter referred to as P50 gene) by immunoscreening using B. gibsoni-infected dog serum. Furthermore, as a result of expressing a protein encoded by the P50 gene (hereinafter referred to as P50 protein), it was confirmed that the expression product had antigenicity and immunogenicity. Therefore, the present invention provides a major antigen of B. gibsoni and a gene encoding the antigen that can be used for the production of a vaccine for diagnosis of B. gibsoni infection and prevention of infection.
[0009]
P50 gene
The present invention provides a gene encoding the major antigenic protein of B. gibsoni. Although this gene can be isolated and identified from a cDNA library as described above, cloning and phosphoramidite using genetic engineering techniques such as general hybridization based on the sequences disclosed in this specification. It may be DNA prepared by a chemical synthesis method such as a method. The form includes cDNA, genomic DNA, and chemically synthesized DNA, but there is no particular limitation.
[0010]
The DNA of the present invention may be single-stranded or may be combined with DNA or RNA having a complementary sequence to form a double strand. The DNA may be labeled with an enzyme such as horseradish peroxidase, a radioisotope, a fluorescent substance, a chemiluminescent substance, or the like.
[0011]
In addition, if the base sequence of the P50 gene is provided, the RNA sequence derived from this, the complementary DNA and RNA sequences, etc. are uniquely determined, so the present invention corresponds to the DNA of the present invention. It is to be understood that DNA and RNA having sequences complementary to the RNA or DNA of the present invention are also provided.
[0012]
Furthermore, the DNA of the present invention also includes DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1. As long as the DNA consisting of the base sequence shown in SEQ ID NO: 1 hybridizes with this under stringent conditions and the protein encoded by the DNA has reactivity with the anti-B. Gibsoni antibody, Sequence variations are allowed. For example, the presence of multiple codons encoding the same amino acid residue by so-called codon degeneracy, various artificial processes such as site-directed mutagenesis, random mutation by mutagen treatment, and mutation / absence of DNA fragments by restriction enzyme cleavage Even if the DNA sequence is partially altered due to loss or ligation, etc., these DNA variants hybridize with the DNA of SEQ ID NO: 1 under stringent conditions, and the anti-B. Gibsoni antibody Any DNA encoding a protein having reactivity is within the scope of the present invention regardless of the difference from the DNA sequence shown in SEQ ID NO: 1.
[0013]
The degree of the above mutation is acceptable if it has a homology of 70% or more, preferably 80% or more, more preferably 90% or more with the DNA sequence shown in SEQ ID NO: 1. In addition, the degree of hybridization is determined according to normal conditions, for example, when the probe is labeled with a DIG DNA Labeling kit (Cat No. 1175033 manufactured by Boehringer Mannheim), a 32 ° C. DIG Easy Hyb solution (Boehringer Mannheim). (Cat No.1603558 manufactured by Cat. No. 1603558) and conditions for washing the membrane in a 0.5 × SSC solution (containing 0.1% (w / v) SDS) at 50 ° C. (1 × SSC is 0.15 M NaCl, 0.015 M It is sufficient that the hybridization with the nucleic acid described in SEQ ID NO: 1 is performed in Southern hybridization with sodium acid.
[0014]
The DNA of the present invention is useful in the preparation of transformed cells, and further in the production of recombinant proteins using the transformed cells.
[0015]
The transformed cells in the present invention can be prepared by applying techniques known to those skilled in the art. For example, various vectors that are commercially available or generally available to those skilled in the art can be used to prepare suitable host cells. It is possible to incorporate the DNA of the present invention.
[0016]
It is also possible to prepare a vaccine preparation containing a recombinant virus incorporating the DNA of the present invention. Such recombinant viruses can also be prepared by applying techniques known to those skilled in the art.
[0017]
P50 protein
The present invention provides a P50 protein that is a major antigen of B. gibsoni encoded by the P50 gene. The deduced amino acid sequence of P50 protein is as shown in SEQ ID NO: 2. This protein is a novel protein having a molecular weight of about 50 kDa and reacts specifically with B. gibsoni-infected dog sera. Furthermore, the deduced amino acid sequence shows no significant homology with any of the previously reported genes of protozoa and other species. Therefore, by using the P50 protein, it is possible to perform a serological diagnosis that does not cross-react with other erythrocytic parasites. Moreover, it is possible to produce and use an anti-B. Gibsoni antibody by using P50 protein as an antigen. Furthermore, it can be used for the production of a vaccine using P50 protein as an immunogen.
[0018]
Amino acid residue side chains that constitute protein components differ in hydrophobicity, charge, size, etc., but are stored in the sense that they do not substantially affect the three-dimensional structure (also referred to as a three-dimensional structure) of the entire protein. Several highly relevant relationships are known. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like. In addition, even if the preservation of the above meaning is impaired, many mutations that do not lose the essential function of the protein are known to those skilled in the art. Furthermore, there are many cases where the same type of protein conserved between different species still retains essential functions even if several amino acids are concentrated or dispersed and deleted or inserted. .
[0019]
Therefore, the protein of the present invention includes an analog of the protein having the amino acid sequence of SEQ ID NO: 2, which retains reactivity with the anti-B. Gibsoni antibody. That is, even if the protein having the amino acid sequence in which the amino acid sequence is deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 is retained as long as it retains reactivity with the anti-B. Gibsoni antibody, the protein of the present invention Is included. Here, “holding reactivity with anti-B. Gibsoni antibody” means that an antigen-antibody reaction can be caused specifically with a monoclonal or polyclonal anti-B. Gibsoni antibody.
[0020]
Such amino acid changes are recognized in nature such as sequence diversity observed between different species or so-called gene polymorphisms, and methods known to those skilled in the art, for example, using a mutagenesis agent such as NTG. It can be artificially generated using site-directed mutagenesis using various mutagenesis methods and various recombinant gene techniques. The amino acid mutation site and number are not particularly limited as long as they retain reactivity with the anti-B. Gibsoni antibody, but the mutation number is usually within several tens of amino acids, preferably within 10 amino acids.
[0021]
antibody
The present invention provides antibodies that bind to the P50 protein. The antibody of the present invention is an antibody that specifically recognizes the entire P50 protein or a partial peptide thereof, and includes monoclonal antibodies and polyclonal antibodies. It belongs to any of the five classes (IgG, IgA, IgM, IgD, IgE) classified as immunoglobulin structures, physicochemical properties and immunological properties, or subclasses depending on the type of H chain. Also good. Furthermore, it may be a fragment such as F (ab ′) 2 when the immunoglobulin is degraded with pepsin, Fab when degraded with papain, or a chimeric antibody. These antibodies can be used to identify P50 gene clones in the immunoscreening of DNA libraries described below. Moreover, it can be used for the production of an antibody column used for purifying P50 protein and for detecting P50 protein in each fraction during purification.
[0022]
[Embodiments of the Invention]
Nucleic acid
As an example of obtaining the DNA of the present invention from a DNA library, a suitable genomic library or cDNA library is screened by a screening method by hybridization, an immunoscreening method using an antibody, etc., and a clone having the target DNA There is a method of growing and then cutting it out using a restriction enzyme or the like. Screening by the hybridization method is performed by labeling a DNA having the nucleotide sequence of SEQ ID NO: 1 or a part thereof with 32P or the like as a probe, and using a known method (for example, Maniatis T. et al. Molecular Cloning, a Laboratory Manual, Cold Spring harbor Laboratory, New York, 1982). As the antibody used in the immunoscreening method, the antibody of the present invention described later can be used. The novel DNA of the present invention can also be obtained by PCR (Polymerase Chain Reaction) using a genomic DNA library or cDNA library as a template. In PCR, a sense primer and an antisense primer are prepared based on the nucleotide sequence shown in SEQ ID NO: 1, and a known method (for example, Michael AI et al., PCR Protocols, a Guide to Methods and Applications) is applied to the DNA library. , Academic Press, 1990) and the like. The DNA library used in the above various methods is based on B. gibsoni-infected cells (see J. Sambrook et al., Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring harbor Laboratory, New York, 1989). A cDNA library can be prepared and used.
[0023]
The recombinant vector having the DNA of the present invention may be in any form such as circular or linear. Such a recombinant vector may have other nucleotide sequences if necessary in addition to the DNA of the present invention. Other base sequences are enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of copy number amplification, base sequences encoding signal peptides, base sequences encoding other polypeptides, These include poly A addition sequences, splicing sequences, replication origins, base sequences of genes that serve as selection markers, and the like.
[0024]
In gene recombination, a translation start codon and a stop codon are added to the DNA of the present invention using an appropriate synthetic DNA adapter, or an appropriate restriction enzyme cleavage sequence is newly generated or deleted in the base sequence. Is also possible. These are within the range of work normally performed by those skilled in the art, and can be arbitrarily and easily processed based on the DNA of the present invention.
[0025]
Moreover, the vector holding the DNA of the present invention may be used by selecting an appropriate vector according to the host to be used. In addition to plasmids, various viruses such as bacteriophage, baculovirus, retrovirus, vaccinia virus, etc. There is no particular limitation.
[0026]
The gene of the present invention can be expressed under the control of a promoter sequence unique to the gene. Alternatively, another suitable expression promoter upstream of the gene of the present invention can be used directly or in place of the promoter sequence unique to the gene. The promoter used in this case may be appropriately selected according to the host and the purpose of expression. For example, when the host is Escherichia coli, the T7 promoter, lac promoter, trp promoter, λPL promoter, etc., and the host is yeast. In some cases, PHO5 promoter, GAP promoter, ADH promoter and the like can be exemplified, but when the host is an animal cell, SV40-derived promoter, retrovirus promoter and the like can be exemplified, but not limited thereto.
[0027]
Methods for introducing DNA into vectors are known (see J. Sambrook et al., Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring harbor Laboratory, New York, 1989). That is, DNA and vector may be digested with appropriate restriction enzymes, and the resulting fragments may be ligated using DNA ligase.
[0028]
A recombinant virus incorporating a nucleic acid molecule of the present invention can be administered to an animal in the form of a vaccine, and such recombinant virus can be expressed in the animal and elicit an immune response against B. gibsoni.
[0029]
Suitable viral vectors include, but are not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses.
[0030]
protein
The protein of the present invention can be prepared by a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes.
[0031]
There are no particular restrictions on the host cells transformed with the recombinant vector of the previous section, and lower cells, insect cells, COS7 that can be used in genetic engineering techniques represented by E. Coli, B. subtils or S. cerevisiae. Many cells such as cells, animal cells typified by CHO cells, and Hela cells can be used for the present invention.
[0032]
Methods for introducing the recombinant vector into the host cell include known methods such as electroporation method, protoplast method, alkali metal method, calcium phosphate precipitation method, DEAE dextran method, microinjection method, method using virus particles, etc. (See Special Issue, Genetic Engineering Handbook, published on March 20, 1991, Yodosha). Any method may be used.
[0033]
In order to produce the protein by genetic engineering, the above-mentioned transformant is cultured, the culture mixture is recovered, and the protein is purified. The transformant can be cultured by a general method. There are various books on the culture of transformants (for example, “Microbial Experiment Method” edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin Co., Ltd., see 1992). .
[0034]
As a method for purifying the protein of the present invention from the culture mixture, an appropriate method can be appropriately selected from methods usually used for protein purification. That is, salting-out method, ultrafiltration method, isoelectric precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, various affinity chromatography such as hydrophobic chromatography and antibody chromatography, chromatofocusing method, adsorption An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and purification may be performed in an appropriate order using an HPLC system or the like as necessary.
[0035]
In addition, the protein of the present invention can be expressed as a fusion protein with another protein or tag (for example, glutathione S transferase, protein A, hexahistidine tag, FLAG tag, etc.). The expressed fusion form can be excised using an appropriate protease (for example, thrombin), and sometimes the protein can be prepared more advantageously. The protein of the present invention may be purified by appropriately combining methods common to those skilled in the art. In particular, when expressed in the form of a fusion protein, it is preferable to employ a purification method characteristic of the form.
[0036]
In addition, a method obtained by a cell-free synthesis method using a recombinant DNA molecule (see J. Sambrook et al., Molecular Cloning, a Laboratory Manual 2nd ed., Cold Spring harbor Laboratory, New York, 1989) This is one of the engineering production methods.
[0037]
As described above, the protein of the present invention can be prepared either alone or in the form of a fusion protein with another type of protein. However, the present invention is not limited to these. It is also possible to convert to For example, processing by various techniques known to those skilled in the art such as various chemical modifications to proteins, binding to polymers such as polyethylene glycol, binding to insoluble carriers, and the like can be considered. In addition, depending on the host used, there is a difference in the presence or absence or the extent of sugar chain addition. Even in such a case, as long as it is a protein having an immunogenicity equivalent to that of the P50 protein, it should still be within the concept of the present invention.
[0038]
antibody
The antibody of the present invention can be obtained by referring to known methods for both polyclonal antibodies and monoclonal antibodies (see, for example, Immunological Experimental Procedures, edited by the Japanese Society for Immunology, published by the Japanese Society for Immunology). Briefly described below.
[0039]
In order to obtain the novel antibody, it is necessary to first administer the protein of the present invention as an immunogen together with an appropriate adjuvant such as Freund's complete adjuvant (FCA) or incomplete adjuvant (FIA) as necessary. Give booster immunizations every 2-4 weeks. After booster immunization, blood is collected to obtain antiserum. The protein of the present invention used as an antigen may be obtained by any method as long as it has a degree of purification that can be used to produce an antibody. The partial polypeptide of the protein of the present invention can also be suitably used as an immunizing antigen. When the polypeptide used as an immunizing antigen is a low molecular weight polypeptide, that is, a polypeptide consisting of about 10 to 20 amino acids, it is combined with a carrier such as keyhole limpet hemocyanin (KLH) as an antigen. Use it. The animal to be immunized can be selected from any animal species that can produce the antibody of interest.
[0040]
The polyclonal antibody can be obtained by purifying the obtained antiserum. Purification may be performed by appropriately combining known methods such as salting out, ion exchange chromatography, affinity chromatography and the like.
[0041]
A monoclonal antibody is obtained as follows. That is, antibody-producing cells such as spleen cells or lymphocytes are collected from the immunized animal and fused with a myeloma cell line or the like by a known method using polyethylene glycol, Sendai virus, electric pulse or the like to produce a hybridoma. Thereafter, a clone producing an antibody that binds to the protein of the present invention can be selected and cultured, and the culture supernatant of the selected clone can be purified. Purification is performed by appropriately combining known methods such as salting out, ion exchange chromatography, affinity chromatography and the like.
[0042]
It can also be obtained from the novel antibody using genetic engineering methods. For example, mRNA is collected from spleen cells, lymphocytes of an animal immunized with the protein of the present invention or a partial polypeptide thereof, or from a hybridoma producing a monoclonal antibody against the protein of the present invention or a partial polypeptide thereof, and cDNA live is used based on this. Make a rally. A clone producing an antibody that reacts with an antigen is screened, the obtained clone is cultured, and the target antibody can be purified from the culture mixture by a combination of known methods.
[0043]
vaccine
An immunologically effective amount of P50 protein or a recombinant virus incorporating the gene of the protein so as to express the protein in vivo is combined with an adjuvant or other pharmaceutically acceptable carrier, and B. gibsoni It is possible to prepare vaccine formulations for the treatment or prevention of infection. “Immunologically effective amount” means an amount administered as part of a single or continuous administration that is effective in treating or preventing B. gibsoni infection; It can be adjusted according to the health condition, vaccine prescription and the like.
[0044]
Examples of the adjuvant include aluminum compounds such as aluminum hydroxide and aluminum phosphate, Freund's adjuvant, and muramyl peptide.
[0045]
The vaccine preparation further includes pH regulators such as sodium phosphate, potassium phosphate, sodium hydroxide and hydrochloric acid, antibiotics such as kanamycin sulfate and erythromycin, lactose, potassium glutamate, D-sorbitol, aminoacetic acid, etc. Stabilizers, isotonic agents such as sodium chloride, and the like can be added.
Anti B.gibsoni Antibody detection method
Since an individual infected with B. gibsoni produces an antibody specific for the P50 protein, it is possible to identify a B. gibsoni-infected dog with high reliability by examining the antibody.
[0046]
In the anti-B. Gibsoni antibody detection method, blood or other biological sample obtained from a test individual is contacted with the purified P50 protein, and a positive individual is identified using an antigen-antibody reaction as an index. Such a test can be performed by a known immunological test method such as indirect fluorescent antibody method (IFA) or passive agglutination method (PA).
[0047]
PCR By law B.gibsoni Infectious DNA Diagnostic method
Diagnosis of B. gibsoni infection is mainly performed by a method of examining blood smears with a microscope (smear microscopy), but has a drawback of low detection sensitivity in chronically infected dogs.
[0048]
DNA diagnosis by PCR can detect very small amounts of protozoan DNA. Therefore, by performing PCR using a primer that is a part of the B.gibsoni gene, it becomes possible to diagnose a B.gibsoni-infected dog that becomes false negative depending on smear microscopy. That is, DNA derived from blood and other samples obtained from a test individual is amplified by PCR using a primer that is a part of the nucleotide sequence shown in SEQ ID NO: 1, and then subjected to electrophoresis to obtain a band derived from the P50 gene. Individuals who have been confirmed can be diagnosed as infected with B. gibsoni.
[0049]
【Example】
EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.
[0050]
Example 1 Identification, cloning and determination of base sequence of B. gibsoni p50 gene
B.gibsoni NRCPD strain [Fukumoto et al., J Parasitol 84: 954-959 (2000)] From infected dog erythrocytes, Acid Guanidinium-Phenol-Chloroform method [Chromczynski et al., Anal Biochem 162: 156-159 (1987)] Total RNA was extracted with. Poly A + RNA was purified from total RNA using mRNA Isolation Kit (Oligotex-dT30, Takara) according to the method recommended by the manufacturer. The construction and immunoscreening of the cDNA library and the plasmidization of the cDNA clone (in vivo Excision) described below were all carried out using Stratagene reagent kits according to the method recommended by the manufacturer. Using about 5 μg of B. gibsoni mRNA as a template, cDNA was synthesized using ZAP-cDNA synthesis kit. The obtained cDNA was size fractionated on a Sephadex CL-2B gel column, then inserted into Uni-ZAP XR Vector arms, and packaged with GigapackIII Gold packaging extract. The packaging product was infected with E. Coli XL1-Blue MRF strain to obtain a library containing about 500,000 cDNA clones.
[0051]
The cDNA library was immunoscreened using B. gibsoni-infected dog sera to obtain two overlapping positive clones. These positive clones were converted into a plasmid (converted to pBluescript SK (+)) by the in vivo Excision method. After the plasmid containing the cDNA fragment was purified with a plasmid purification kit (Qiagen), PCR was performed using the Dye Primer Cycle Sequencing Kit (Perkin Elmer) according to the method recommended by the manufacturer. PCR products were analyzed using ABI PRISM 377 DNA sequencer (Perkin Elmer), and the nucleotide sequence of the cDNA fragment was determined. As a result, two clones were found to be the same gene. Among them, a clone having a long fragment was used for further analysis. The total length of the cDNA was 1924 bp, and it was confirmed that it had an open reading frame (ORF) of 1401 bp (SEQ ID NO: 1). The deduced protein consisted of 466 amino acids, and the deduced molecular weight was 50 kDa (SEQ ID NO: 2). Hereinafter, this gene is referred to as P50 gene. A homology search of the amino acid sequence predicted from the P50 gene using the BLAST method of the National Center for Biotechnology Information showed significant homology with any of the protozoa and any other species reported so far. There wasn't.
[0052]
Example 2 Construction of a recombinant vector plasmid for expressing B. gibsoni P50 protein in E. coli
The pBluescript plasmid into which the B. gibsoni P50 cDNA fragment was inserted was digested with restriction enzymes EcoRI and XhoI to excise the P50 cDNA fragment and inserted into the EcoRI and XhoI sites of the E. coli expression vector pGEMEX-2 (Promega). The recombinant plasmid pGEMEX-2 / P50 was purified using a plasmid purification kit (Qiagen). The plasmid pGEMEX-2 / P50 is deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number FERM BP-7533.
[0053]
Example 3 Expression of B. gibsoni P50 protein in E. coli
E. coli JM109 (DE3) strain (Promega) was transformed with the recombinant plasmid obtained in Example 2, and cultured at 37 ° C. in LB medium containing ampicillin. The OD value (600 nm of the culture solution) ) Reached 0.3 to 0.5, IPTG was added to a final concentration of 0.5 mM, and the culture was further continued at 37 ° C. for 4 hours. Recombinant protein expression was confirmed by Coomassie staining after electrophoresis [Laemmli et al., Nature 227: 680-685 (1970)] on a 10% SDS-polyacrylamide gel. As a result, the expression of a recombinant protein of about 85 kDa was confirmed, and it was confirmed that it was a fusion protein of gene10 leader protein (35 kDa) and B. gibsoni P50 protein (50 kDa) (FIG. 1).
[0054]
Example 4 Purification of recombinant P50 protein expressed in E. coli and production of antibodies against the purified protein
The recombinant P50 protein expressed in E. coli by the method described in Example 3 was purified according to the method recommended by Promega. The electrophoresis image of the purified recombinant P50 protein is shown in FIG. Next, to prepare an antibody against the purified recombinant P50 protein, 200 μL of a solution containing 100 μg of recombinant P50 protein and 200 μL of Freund's complete adjuvant (FCA; Difco) were mixed and emulsified with BALB / c Mice (8 weeks old, female) were inoculated intraperitoneally. At 2 weeks and 4 weeks after inoculation, 100 μg of recombinant P50 protein mixed with FCA and emulsified were additionally inoculated. Blood was collected 2 weeks after the final inoculation, and the serum was stored at -20 ° C.
[0055]
Example 5 Identification of native (native) P50 protein by immunoblotting
Using the anti-recombinant P50 protein mouse serum obtained in Example 4, the natural product P50 protein was identified by immunoblotting [Towbin et al., Proc Natl Acad Sci USA 76: 4350-4354 (1979)]. . As shown in FIG. 2, a specific band of 50 kDa was detected in B. gibsoni-infected erythrocyte lysate. The molecular weight of the native P50 protein was similar to the estimated theoretical value (50 kDa).
[0056]
Example 6 Reactivity of immune sera to recombinant P50 protein
The reactivity of B. gibsoni-infected dog sera to recombinant P50 protein was examined by immunoblotting. As shown in FIG. 3, it was confirmed that the recombinant P50 protein reacted strongly with B. gibsoni-infected dog serum. In contrast, no reaction between normal dog serum and recombinant P50 protein was observed.
[0057]
This indicates that the recombinant P50 protein is useful as a diagnostic antigen for B. gibsoni infection. It was also shown to be useful as a vaccine using P50 as an immunogen.
[0058]
Example 7 Antigenicity of recombinant P50 protein to dogs
The antibody production induction of the recombinant P50 protein shown in Example 4 to dogs was examined. Purified recombinant P50 protein alone or a mixture of this protein plus aluminum gel as an adjuvant was inoculated subcutaneously or intramuscularly into 3 month old beagle dogs and boosted at 4 weeks after the first inoculation. Blood was collected 4 weeks after the additional inoculation. Recombinant baculovirus AcP50 expressing the P50 gene was prepared, and AcP50-infected Sf9 cells were treated with Triton X-100. The soluble fraction was used as an antigen and reacted with the serum, and an antibody bound to the antigen was detected by an enzyme antibody method (ELISA method). As a result, as shown in Table 1, antibodies against the recombinant P50 protein were found in the sera of all the immunized groups, but no antibodies were found in the sera of dogs in the non-inoculated control group.
[0059]
From this result, induction of antibody production against this protein was confirmed by inoculating the dog with the recombinant P50 protein, and the usefulness of the recombinant P50 protein as an anti-B. Gibsoni vaccine was demonstrated.
[0060]
[Table 1]

[0061]
【The invention's effect】
The gene is a gene encoding a novel major antigenic protein P50 protein of B. gibsoni, and since the protein has antigenicity to dogs, it can be used as an anti-B. Gibsoni vaccine. In addition, by using the present protein or a fragment thereof or a base sequence encoding the present protein as a probe, diagnosis of B. gibsoni protozoan infection, which has conventionally been difficult to distinguish from closely related species, can be performed more specifically.
[0062]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows an electropherogram showing a recombinant P50 protein which is an expression product of the present invention.
Lane 1: molecular weight marker, lane 2: purified recombinant P50 protein, lane 3: purified gene 10 protein
FIG. 2 shows an immunoblot image showing detection of native P50 protein by recombinant P50 protein immune serum.
Lane 1: B.gibsoni-infected red blood cells, Lane 2: normal dog red blood cells
FIG. 3 shows an immunoblot image showing the reactivity between recombinant P50 protein and B. gibsoni-infected dog serum. Lane 1: purified recombinant P50 protein, lane 2: purified gene 10 protein

Claims (11)

The following protein (a) or (b) or a peptide fragment thereof ;
(A) a protein having the amino acid sequence shown in SEQ ID NO: 2 ;
(B) a protein having an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having reactivity with an anti-Babiesia gibsoni antibody , or Peptide fragment reactive with anti-Babesia gibsoni antibody .   The protein according to claim 1, which has a protective immunity-inducing activity against B. gibsoni.   A nucleic acid molecule having a nucleic acid molecule encoding the protein or polypeptide according to claim 1 or 2 or a complementary base sequence thereof.   The nucleic acid molecule of Claim 3 which has a base sequence shown to sequence number 1, or its complementary base sequence.   A recombinant vector comprising the nucleic acid molecule according to claim 3 or 4.   A recombinant virus incorporating the nucleic acid molecule according to claim 3 or 4.   A transformed cell transformed with the recombinant vector according to claim 5.   An antibody against the protein or polypeptide according to claim 1.   A B. containing the protein of claim 2. A vaccine for treating or preventing gibsoni infection.   A vaccine for the treatment or prevention of B. gibsoni infection comprising the recombinant virus according to claim 6. A method for diagnosing B. gibsoni infection in dogs using a nucleic acid encoding a protein having the amino acid sequence shown in SEQ ID NO: 2 as a marker.

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