JP2014000016A - Novel antigenic protein of erysipelothrix rhusiopathiae, gene thereof, recombinant vector, and the use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective, economical, high-safety vaccine against erysipelothrix rhusiopathiae.SOLUTION: The vaccine relates to a recombinant protein having a homology of 90% or more with a specific amino acid sequence and protective immunity-inducing activity against erysipelothrix rhusiopathiae, a gene which encodes the recombinant protein and is composed of DNA which encodes a protein having a homology of 90% or more with another specific base sequence and protective immunity-inducing activity against erysipelothrix rhusiopathiae, and an erysipelothrix rhusiopathiae vaccine for humans or non-human animals, containing the recombinant protein.

Description

  The present invention relates to an antigen for a vaccine against swine erysipelas. Specifically, the present invention relates to a novel antigenic protein of swine gonococcus, its gene, and a recombinant vector, and their use as a vaccine for protecting against infection of swine gonococci.

  Swine erysipelas is a zoonotic disease caused by infection with swine erysipelas (Eryciperothrix rhusiopathie). It has been confirmed that the porcine erysipelas infects pigs, wild boars, mammals including humans, and birds. Among them, as the name suggests, it is famous as a bacterium that causes swine disease, and swine erysipelas occurs in pig farms around the world. The course of swine erysipelas is very acute and has a high fatality rate, and if it takes a chronic course, it may cause arthritis and become a colonized pig. If a shipped pig becomes positive in the slaughter test, the individual is totally discarded and the economic loss is extremely large. In addition, it is known that when porcine erysomycetes infects humans, it causes sepsis and arthritis, and porcine erysococcus is regarded as an important bacterium not only for animal health but also for public health.

  Vaccines are effective in preventing swine erysipelas. In Japan, attenuated live and inactivated vaccines resistant to acriflavine are commercially available from various manufacturers and widely used. This live attenuated vaccine is a bacterium that has been attenuated by subculturing a virulent strain on an agar medium supplemented with acriflavine, and it is said that immunity for 6 months or more can be sustained by a single inoculation.

  On the other hand, inactivated vaccines are inactivated antigens of whole cells, and are widely used in Japan as simple vaccines as well as mixed vaccines (mixed 2 and 3 types). Unlike live vaccines, in order to induce strong immunity, an adjuvant is added for the purpose of immunization and requires two inoculations. Problems with using inactivated vaccines include safety and cost issues such as fever during inoculation, but for this reason, only the components involved in defense are identified, and vaccines that consist only of those components (component vaccines) It is desirable to provide As a technique similar to this, there is a protective antigen comprising a surface protective antigen polypeptide derived from serotype 18 bacteria and a partial polypeptide (SpaCΔC) from which the 237 amino acids present at the carboxyl terminus have been removed (see Patent Document 1).

  In addition, the present inventors, Shimochi, Ogawa, et al., Have deciphered the whole genome of swine erysipelas, and newly reported SpaA and RspA antigens as antigens involved in the protection of swine erysipelas (Non-patent Document 1). ). Among them, the SpaA antigen is a major protective antigen on the surface of the cell body, and it has been confirmed that immunization with this protein together with an adjuvant can induce protection in mice and pigs.

JP 2006-25659 A

Journal of Bacteriology, 193: 2959-2971,2011

  The present invention relates to a novel swine venom vaccine that can effectively prevent infection of swine gonococci in humans or non-human animals, can be produced economically, and is highly safe. , A recombinant protein that can be used in the vaccine, and a method for producing the same, a gene encoding the recombinant protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, and a pig for human or non-human animals The purpose is to provide a bactericidal vaccine.

  The present inventors pay attention to the fact that in Non-Patent Document 1, SpaA contains a repetitive sequence (GW module) of about 20 amino acid sequences starting with glycine and tryptophan at the C-terminus, and this GW module is Streptococcus pneumoniae. It was predicted to be a choline-binding protein found in (Streptococcus pneumoniae). And, like the Streptococcus pneumoniae, like Streptococcus pneumoniae, it has a plurality of choline-binding proteins, and from the whole genome sequence of the S. pneumoniae Fujisawa strain, a protein antigen containing a repetitive amino acid sequence containing a GW module at the C-terminus When the gene was searched, a choline-binding protein whose function was unknown in Non-Patent Document 1 was found, a recombinant protein was prepared based on this base sequence, and it was analyzed whether protection was induced in the inoculated animal. As a result, it was confirmed for the first time that the recombinant protein of ERH_0768 had a vaccine effect, and the present invention was completed.

That is, the gist of the present invention is as follows.
[1] A recombinant protein having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 1 and having protective immunity induction activity against swine red beetle,
[2] The recombinant protein comprising the amino acid sequence shown in SEQ ID NO: 1,
[3] a gene encoding the recombinant protein,
A gene comprising a DNA encoding a protein having 90% or more homology with the base sequence shown in SEQ ID NO: 2 and having protective immunity-inducing activity against S. elegans;
[4] The gene comprising DNA consisting of the base sequence shown in SEQ ID NO: 2,
[5] a recombinant vector containing the gene,
[6] A transformant containing the recombinant vector,
[7] A method for producing the recombinant protein comprising the steps of culturing the transformant in a medium and collecting the recombinant protein from the obtained culture,
[8] Swine erysipelas vaccine for human or non-human animals containing the recombinant protein,
[9] A porcine erysipelas vaccine for human or non-human animals using the recombinant vector,
[10] The above-mentioned swine venom vaccine for human or non-human animals further comprising adjuvant
[11] The present invention relates to the aforementioned porcine erysipelas vaccine for humans or non-human animals for pigs.

ADVANTAGE OF THE INVENTION According to this invention, the recombinant protein which can be utilized as a swine venom vaccine for humans or a non-human animal is provided. This recombinant protein can be produced in large quantities by non-pathogenic recombinant bacteria using genetic recombination technology, and vaccines using this recombinant protein are not necessary for vaccine activity unlike commercially available inactivated vaccines Because it is a component vaccine that can reduce various components, it is highly safe and cheaper than a vaccine that uses swine erysipelas whole cells.
Moreover, a DNA vaccine or a virus vaccine can also be produced using a recombinant vector containing a gene encoding the recombinant protein.

FIG. 1 shows the nucleotide sequence of the ERH_0768 gene product expressed as a recombinant antigen: the underline is derived from pQE-30, and the others are the nucleotide sequences of ERH_0768. FIG. 2 shows the amino acid sequence of the ERH_0768 gene recombinant antigen: underlined; derived from pQE-30, others indicate the sequence of ERH_0768. FIG. 3 shows an electropherogram of the ERH_0768 gene product purified in Example 1. FIG. 4 shows the number of surviving mice after challenge with a virulent swine gonococcal strain performed in Example 2.

1. TECHNICAL FIELD The present invention relates to a recombinant protein having at least 90% homology with the amino acid sequence shown in SEQ ID NO: 1 and having protective immunity-inducing activity against swine erysipelas.

The amino acid sequence shown in SEQ ID NO: 1 is composed of 597 amino acids. The 16 amino acids on the N-terminal side are derived from the expression vector pQE-30 (QIAGEN). The following 581 amino acids are sequences derived from ERH_0768 (CDS T01525), which is a sequence obtained by removing 25 amino acids on the N-terminal side, which is a signal sequence, from the entire amino acid sequence.
It should be noted that the entire amino acid sequence and the entire base sequence of ERH_0768 can be confirmed with a known database such as KEGG (Kyoto Encyclopedia of Genes and Genomes).

In the present invention, “having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1” means “protein blast” (http://blast.ncbi.nlm.nih.gov/Blast.cgi) on the NCBI website. ) And the like, and the homology with the amino acid sequence shown in SEQ ID NO: 1 is examined, it means 90% or more homology.
Specifically, an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 can be mentioned. The position of the sequence to be subjected to these mutations is not particularly limited, but may be any position as long as it does not lose the protective immunity induction activity against swine erysipelas described below in addition to being within the above-mentioned homology range. In the present invention, when an expression vector other than pQE-30 is used, an amino acid sequence in which the amino acid sequence on the N-terminal side of SEQ ID NO: 1 is changed is also included in the “amino acid sequence shown in SEQ ID NO: 1”. Further, it is assumed that an amino acid sequence having all of the amino acid sequence shown in SEQ ID NO: 1 has 100% homology.

  In the present invention, “having protective immunity induction activity against swine erysipelas” means nonimmune when a swine erysipelas highly virulent strain (Fujisawa strain) is infected by the technique as described in Example 2 described later. This means that the lethal activity is significantly reduced as compared with the mice.

  The recombinant protein of the present invention is preferably a protein comprising the amino acid sequence shown in SEQ ID NO: 1 from the viewpoint of easy production and excellent protective immunity induction activity.

  The recombinant protein of the present invention can be prepared from a gene encoding the recombinant protein.

2. Gene The gene encoding the recombinant protein is a gene comprising a DNA encoding a protein having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 and having protective immunity-inducing activity against Porphylococcus pneumoniae. Can be mentioned.
The basic acid sequence shown in SEQ ID NO: 2 is composed of 1794 bases. The 48 base sequences at the 5 ′ end are derived from the expression vector pQE-30 (QIAGEN). The subsequent 1746 amino acids are a sequence derived from ERH_0768, which is a sequence obtained by removing 75 bases on the 5 ′ end side, which is a signal sequence, from the entire base sequence.

In the present invention, “having 90% or more homology with the nucleotide sequence shown in SEQ ID NO: 2” means that 90% or more homology as a result of homology search using “nucleotide blast” or the like on the NCBI website. Is meant to indicate Specifically, a base sequence in which one or several bases have been deleted, substituted or added in the base sequence of SEQ ID NO: 2 can be mentioned. The position of the sequence to be subjected to these mutations is not particularly limited, but in addition to being within the above-mentioned homology range, the protein produced by the transformant induces protective immunity against the porcine erysococcus as described above. Any position may be used as long as it does not lose the activity. For example, a gene containing DNA consisting of the base sequence shown in SEQ ID NO: 2 can be mentioned. In the present invention, when an expression vector other than pQE-30 is used, the base sequence in which the base sequence on the 5 ′ end side of SEQ ID NO: 2 is changed is also included in the “base sequence shown in SEQ ID NO: 2”.
Further, it is assumed that the base sequence having all the base sequence shown in SEQ ID NO: 2 has 100% homology.

Nucleotide sequence shown in the SEQ ID NO: 2, for example, can be obtained by combining a conventional method, it can also be obtained in the manner described in Example 1 below.
Moreover, with respect to the obtained base sequence, one or several bases may be deleted, substituted, or added based on a known method as necessary.

3. Recombinant vector The recombinant vector of the present invention can be obtained by linking (inserting) the gene of the present invention to a known vector such as a plasmid. The recombinant vector is not particularly limited as long as it can replicate in a host, and examples thereof include plasmid DNA and phage DNA.

  Examples of the plasmid DNA include plasmids derived from E. coli (eg, pBR322, pBR325, pUC18, pUC119, pTrcHis, pBlueBacHis, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, etc.), and plasmids derived from yeast (eg, YEp13, YEp24, YCp50, pYE52, etc.), plasmids for plant cell hosts (pBI221, pBI121) and the like, and phage DNAs include λ phage and the like. Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can be used.

  In order to insert the gene of the present invention into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector. Adopted. The gene of the present invention needs to be introduced by linking to a promoter corresponding to the host in such a manner that the gene can function. Here, the “functional aspect” means that the gene of the present invention arranged downstream thereof is appropriately expressed in the host and exerts its function by the promoter activity. The type of promoter used is appropriately determined depending on the host cell, and details thereof will be described in the next section. The vector of the present invention includes a promoter, the gene of the present invention, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker (for example, a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, etc. ), A ribosome binding sequence (SD sequence) and the like.

In the present invention, in addition to a constitutively expressed promoter, a promoter that is activated in a condition-specific (for example, tissue-specific or environment-specific) can be preferably used. By using such a promoter, the gene of the present invention can be expressed in a condition-specific manner. For example, the recombinant vector can be used as a DNA vaccine, a virus vaccine or a bacterial vaccine. In any case, it is desirable to select a promoter that functions in the non-human animal to be administered. The promoter used may be of any base sequence, as long as it can function effectively as a promoter in a transcription system possessed by a non-human animal to be administered regardless of whether it is synthetic or natural. Virus-derived DNA and eukaryotic or prokaryotic DNA can be used in the present invention as long as the above conditions are satisfied.
In the DNA vaccine, the DNA incorporating the recombinant vector is used as a vaccine. In the virus vaccine, a virus containing the recombinant vector is used as a vaccine. In the bacterial vaccine, a bacterium containing the recombinant vector is used as a vaccine. An appropriate DNA, virus, or bacterium may be appropriately selected for the non-human animal to be treated and prepared according to a known technique. In addition, the bacteria used for the bacterial vaccine can be the same as those used as hosts for the transformants described below.

4). Transformant (host cell)
The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. The host is not particularly limited as long as it can express the gene of the present invention. For example, Escherichia coli, Bacillus subtilis (Bacillus subtilis) and the like, Pseudomonas putida (Pseudomonas putida) Pseudomonas, Rhizobium meliloti (Rhizobium meliloti) Rhizobium genus (Rhizobium) Saccharomyces cerevisiae, Schizosaccharomyces pombe and other yeast cells, plant cells established from Arabidopsis thaliana, tobacco, corn, rice, carrots, etc., animal cells such as protoplasts, COS cells, and CHO cells, or Sf9 And insect cells such as Sf21.
In addition, when the host into which the recombinant vector of the present invention has been introduced is used as a vaccine, for example, viral vaccines include adenovirus, poxvirus, herpesvirus,
Examples of viruses and bacterial vaccines include swine erysipelas, lactic acid bacteria, and Salmonella.
These hosts may be appropriately selected according to the purpose.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium, and at the same time is composed of a promoter, a ribosome binding sequence, a gene of the present invention, and a transcription termination sequence. Is preferred. Moreover, the gene which controls a promoter may be contained. Examples of E. coli include E. coli HMS174 (DE3), K12, DH1, and the like, and examples of Bacillus subtilis include Bacillus subtilis MI 114, 207-21. A promoter is not particularly limited as long as it can be expressed in said host in such as E. coli, for example, trp promoter, lac promoter, P _L promoter, such as P _R promoter, promoters derived from Escherichia coli or phage and the like . An artificially designed and modified promoter such as a tac promoter may be used. The method for introducing a recombinant vector into bacteria is not particularly limited. For example, a method using calcium ions [Cohen, SN et al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972) ] And electroporation methods.

  When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris and the like are used. The promoter is not particularly limited as long as it can be expressed in yeast.For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. Can be mentioned. The method for introducing a vector into yeast is not particularly limited. For example, the electroporation method [Becker, DM et al .: Methods. Enzymol., 194: 182-187 (1990)], the spheroplast method [Hinnen, A. et al .: Proc. Natl. Acad. Sci., USA, 75: 1929-1933 (1978)], lithium acetate method [Itoh, H .: J. Bacteriol., 153: 163-168 (1983)] Etc.

  When plant cells are used as hosts, for example, cells established from rice, corn, wheat, Arabidopsis thaliana, tobacco, carrots, etc., or protoplasts prepared from the plants are used. In this case, the promoter is not particularly limited as long as it can be expressed in plants, and examples thereof include cauliflower mosaic virus 35S RNA promoter, rd29A gene promoter, and rbcS promoter. Examples of the method for introducing a recombinant vector into a plant include an indirect introduction method such as an Agrobacterium infection method, and a direct introduction method such as a particle gun method, a polyethylene glycol method, a liposome method, and a microinjection method. Alternatively, plant cell transformation using the Agrobacterium infection method may be used.

  When a virus is used as a host, for example, a promoter derived from a virus such as CMV, RSV or SV40 is used.

5. Production of Recombinant Protein The recombinant protein of the present invention is obtained by culturing the above-described transformant (host cell) in an appropriate medium, and collecting a protein having protective immunity-inducing activity against sweptococcus pneumoniae from the culture. Can be obtained. The transformant of the present invention may be cultured according to a conventional method. For example, in the case of a transformant having a microorganism such as Escherichia coli or yeast as a host, a natural medium containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism, and capable of efficiently culturing the transformant, Alternatively, it may be cultured in a synthetic medium. In addition, when plant cells are used as a host, the cells may be cultured in a plant cell medium supplemented with vitamins such as thiamine and pyridoxine.

  As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol are used. As the nitrogen source, ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like are used. As the inorganic substance, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used.

  In the medium, antibiotics such as ampicillin and tetracycline may be added to the medium as necessary. When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with an expression vector using Lac promoter, cultivate a microorganism transformed with isopropyl-β-D-thiogalactopyranoside (IPTG) or the like with an expression vector using trp promoter. Sometimes indoleacrylic acid (IAA) or the like may be added to the medium.

  The culture is usually performed at about 30 to 37 ° C. for about 6 hours to 3 days under aerobic conditions such as shaking culture or aeration and agitation culture. During the culture period, the pH is maintained at about 7.0 to 7.5. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. When the recombinant protein of the present invention is produced in cells or cells after culturing, the recombinant protein is extracted by disrupting the cells or cells. When the recombinant protein of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, general biochemical methods used for isolation and purification of recombinant proteins, such as ammonium sulfate precipitation, SDS-PAGE, gel chromatography, ion exchange chromatography, affinity chromatography, etc., are used alone or in appropriate combination. Thus, the recombinant protein of the present invention can be isolated and purified from the culture.

6). Porcine Reptococcus Vaccine The recombinant protein of the present invention can be used as an antigen for a vaccine (hereinafter referred to as porcine vulgaris vaccine) for preventing infection with sweptococcus in humans or non-human animals. Therefore, the swine venom vaccine of this invention contains the said recombinant protein. However, when the recombinant protein is used as a vaccine as it is, the production of antibodies and the protective effect against infection are considered to be weak. Therefore, for example, by formulating it in appropriate combination with a pharmacologically acceptable carrier or medium, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, etc. Therefore, it is necessary to increase the production of antibodies against the recombinant protein and the protective effect against infection. Addition of various immunostimulants is also effective.

In addition, other forms of swine erysipelas vaccine include DNA vaccines, virus vaccines, and bacterial vaccines using the recombinant vectors containing the gene encoding the recombinant protein.
The method for preparing the DNA vaccine, virus vaccine, bacterial vaccine or the like is not particularly limited. DNA vaccines, virus vaccines, bacterial vaccines, etc. should be formulated in combination with sterilized water, physiological saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, etc. as appropriate in the case of the above recombinant protein. Can do.

Moreover, you may mix | blend an adjuvant with the said porcine erysipelas vaccine. Examples of the adjuvant to be added include inorganic substances such as aluminum salts, microorganisms or microorganism-derived substances (BCG, muramyl dipeptide, pertussis, pertussis toxin, cholera toxin, etc.), surfactant substances (saponins, deoxygens). Cholic acid, etc.), emulsions of oily substances (mineral oil, vegetable oil, animal oil), etc., and these can be used alone or in combination.
The vaccine of the present invention can obtain an excellent protective effect and high safety against swine erysipelas by blending this adjuvant.

  The vaccine of the present invention is intended to be administered to human or non-human animals. Examples of non-human animals include pigs and wild boars, mammals including humans and mice, and birds.

  Administration of the vaccine of the present invention to humans or non-human animals is, for example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular or oral administration to those skilled in the art. It can be performed by a known method. The dose varies depending on the body weight and age of human and non-human animals, the administration method, the purpose of use, etc., but those skilled in the art can appropriately select an appropriate dose.

  The action of the vaccine of the present invention includes an action that inhibits the onset of infectious diseases by prophylactic administration to animals that have not been infected with swine erysipelas species or animals that are in a pre-onset state after infection. Is included. Inhibition of this onset includes 1) an action that prevents the onset itself, 2) an action that delays the onset, 3) an action that reduces symptoms after the onset, 4) an action that accelerates healing after the onset, 5) from the individual who developed the disease, etc. The action which inhibits the infectious power to the individual | organism | solid of is included.

Example 1 Production of Recombinant Protein A protein containing a repetitive amino acid sequence containing the GW module at the C-terminus in the entire genome sequence of S. pyogenes (accession number AP012027) was identified to identify ERH_0768. The ERH_0768 gene was amplified by PCR using primers 768F (SEQ ID NO: 3: CCCGAGCTCGAGGAGCGAAAACCGAGAGAA) and 768R (SEQ ID NO: 4: CCCAAGCTTCTATTTTTTTAGTGCTCCTTG) using the genomic DNA of the porcine erysipelas Fujisawa strain as a template. This amplified product was digested with restriction enzymes Sac I and Hind III, ligated into pQE30 vector (Qiagen) digested with the same restriction enzymes, and then cloned. The PCR, ligation and cloning were performed based on known methods. Further, the base sequence was determined by the Sanger method, and the sequence shown in FIG. This recombinant protein contains 6 consecutive histidine sequence tags on the N-terminal side as shown in FIG. 2 and SEQ ID NO: 1, and 25 amino acids on the N-terminal side are signal sequences from the entire amino acid sequence of ERH_0768. It is a recombinant protein of about 70 kDa consisting of the removed amino acid sequence of ERH_0768 (see FIG. 3).

(Example 2) Efficacy test in mice The recombinant protein obtained in Example 1 was evaluated for its effectiveness as a recombinant vaccine using a mouse swine infection model as follows.

(Materials and methods)
Test animals: 20 ddy mice of 4 weeks of age were divided into 10 vaccine groups and 10 control groups.
Preparation of vaccine: ERH_0768 gene product diluted with PBS to a final concentration of 0.2 mg / ml, 1 volume and 1/3 volume of ISA-25 (SEPPIC) mixed and emulsified were used as the vaccine.
Vaccination method: Each vaccine group was inoculated 0.1 ml into the lower limb muscle twice at 2-week intervals. The control group was not inoculated.
Attack material: A bacterial solution obtained by overnight culturing swine erysipelas strain (Fujisawa strain) was used as an attack material.
Attack method: Two weeks after the final immunization, 0.1 ml of attack material (1 × 10 ⁵ CFU / ml) was inoculated subcutaneously into the lower limbs.
Observation and effect determination: Clinical symptoms were observed for 10 days after the attack.

The result is shown in FIG.
In the control group, the death of the mice was observed from the second day after the challenge, and all the heads died on the fourth day. On the other hand, in the vaccine immunization group, the death of the mice was observed from the fifth day, and the survival rate at the end of the observation was 30%.
From these results, it was confirmed that the mice immunized with the ERH_0768 gene product exhibited a protective effect against lethal activity due to the attack of swine erysipelas highly toxic strain (Fujisawa strain).

  In addition, as described above, since the protective effect was confirmed in mice with the possibility of infection with swine venom, it also has a protective effect on pigs, wild boars and other mammals, birds, and humans that are the host of swine venom. I can expect.

Claims (11)

  A recombinant protein having a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 1 and having protective immunity-inducing activity against Porphylococcus pneumoniae.   The recombinant protein according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 1. A gene encoding the recombinant protein according to claim 1 or 2,
A gene comprising a DNA encoding a protein having a homology of 90% or more with the base sequence shown in SEQ ID NO: 2 and having protective immunity induction activity against Porphylococcus pneumoniae.   The gene according to claim 3, comprising DNA consisting of the base sequence shown in SEQ ID NO: 2.   A recombinant vector comprising the gene according to claim 3 or 4.   A transformant comprising the recombinant vector according to claim 5.   The method for producing a recombinant protein according to claim 1 or 2, comprising a step of culturing the transformant according to claim 6 in a medium and collecting the recombinant protein from the obtained culture.   A swine erysipelas vaccine for human or non-human animals comprising the recombinant protein according to claim 1 or 2.   A swine erysipelas vaccine for human or non-human animals using the recombinant vector according to claim 5.   Furthermore, the swine venom vaccine for humans or non-human animals of Claim 8 or 9 containing adjuvant.   It is for pigs, The swine venom vaccine for humans or non-human animals in any one of Claims 8-10.