JP2006025659A - New polypeptide having prevention activity against erysipelothrix rhusiopathiae infection derived from serum type 18 being another strain of erysipelothrix, gene thereof and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a recombinant polypeptide that is used as a main component for a medicine composition such as a vaccine for preventing infectious diseases of swine erysipelas and exhibits high safety and excellent effectiveness when immunized against a pig and to provide a method for producing the same. <P>SOLUTION: A prevention antigen exhibiting high safety and excellent effectiveness to pigs is obtained by preparing SpaC (antigen polypeptide of surface layer prevention derived from serum type 18 belonging to other strains of Erysipelothrix) and a partial polypeptide (SpacCΔC) obtained by removing 237 amino acid existing at its carboxy terminal. The component is mass expressed as a recombinant polypeptide and readily prepared by the production method. The SpaC and SpaCΔC polypeptide are effectively used as a vaccine component for preventing infectious diseases of swine erysipelas and as an antigen for assaying an antibody against swine erysipelas. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel polypeptide having anti-infection activity derived from serotype 18 which is another bacterium of the genus Erysperoperix, a gene encoding the same, and an antibody against the polypeptide. More specifically, the present invention relates to a polypeptide as an immunogen for providing a pharmaceutical composition such as a vaccine for preventing swine erysipelas when immunized and a method for producing the same.

  Swine erysipelas is a contagious disease of pigs caused by swine erysipelas. Affected pigs have a septic form that suddenly died acutely, a subacute form with characteristic urticaria, and no obvious clinical symptoms, but often found in slaughterhouses as a problem endocarditis Various pathological conditions such as type and arthritic type are shown. High mortality is a problem in acute cases. On the other hand, in the chronic type, in addition to a decrease in productivity due to growth delay or the like, if this disease is found in a slaughterhouse, the carcass is entirely discarded, so the economic damage is great. In addition, since there are cases where this bacterium infects humans and causes erysipelas, it is considered to be a problem from the viewpoint of public health as a causative bacterium of zoonotic diseases.

  Swine erysipelas are currently classified into 26 types of serotypes and N-types lacking the antigens, based on the serological types of thermostable antigens in peptidoglycans present on the cell walls. In recent years, Takahashi et al. (Non-Patent Document 1) applied a classification method using DNA-DNA homology and suggested that these serotypes can be divided into four species. That is, serotypes 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, 21 and N-type are designated as Erysipethrix rhusiopathiae (hereinafter abbreviated as “Er”). Serotypes 3, 7, 10, 14, 20, 22, and 23 are used for Erysiperothrix tonillarum (hereinafter abbreviated as “Et”), and serotype 13 is used for other Erysiperothrix species 1 18 was classified into the other species of the genus Elysperotrix. From the epidemiological survey, Er is the bacterial species with strong pathogenicity in pigs, and among them, type 1a, 1b and type 2 have a high separation rate from swine erysipelas cases. However, according to the results of the infection experiment, all the serotypes of Er are considered to be pathogenic to pigs.

  In order to prevent this disease, live vaccines have been used in Japan for a long time and have contributed to the prevention of pressure. The live vaccine strain is a attenuated strain of the Koganei strain, which is a serotype 1a highly toxic strain, which is subcultured for a long time in a medium supplemented with acriflavine. Recently, however, cases of chronic swine erysipelas in slaughterhouses have been increasing, and the following weak points of live vaccines have been pointed out. First, since live vaccines inhibit the vaccine take by using migrating antibodies and antibacterial agents, there are cases where the vaccine effect cannot be fully exerted on some farms. Secondly, with recent changes in breeding habits, many animals with good hygiene, such as SPF pigs, have been kept. When such a pig is inoculated with this vaccine, it depends on the pathogenicity of the vaccine strain itself. Some cases have developed.

  In 1997, in order to overcome such weaknesses of swine erysipelas vaccine, the manufacture of an inactivated vaccine was approved and used for the first time in Japan. In this way, inactivated vaccines have many advantages over live vaccines, but the cost of manufacturing Er by culturing Er itself and when mixed with other vaccines used in swine There is a problem that it is easy to cause an interference action with the ingredients of this, and it is difficult to develop a mixed preparation. Therefore, a component vaccine obtained by extracting and purifying only an antigen polypeptide related to infection protection from Er is most desirable. From the viewpoint of cost, production using a recombinant is preferable.

  Regarding the antigen that induces infection protection of this bacterium, a polypeptide present on the surface of the microbial cell called SpaA having a molecular weight of 65-70 kDa is known. The gene (spaA) derived from Er serotype 2 was analyzed by Makino et al. (Non-patent document 2), and the serotype 1 gene was analyzed by Shimoji et al. (Non-patent document 3) and Imada et al. (Non-patent document 4). The sequence has been revealed. Mice and pigs immunized with SpaA polypeptide expressed by recombinant E. coli have been shown to be protected from lethal attack by viable Er. Therefore, it is considered that this SpaA polypeptide can be an important component of a preventive vaccine for swine erysipelas. On the other hand, the protective activity of this SpaA polypeptide has been demonstrated only against Er serotypes 1 and 2, and has the same protective activity against attacks of other Er serotypes resident in the field. Whether or not to indicate is unknown. As the antigen polypeptide used for the vaccine, those having a broad and high level of protective activity against various Er serotypes are more preferable.

According to the report of Makino et al., The spaA gene is present in 11 serotypes of the 16 serotypes of Er (1a, 1b, 2, 5, 8, 9, 12, 12, 15, 16, 17 and N) and the rest Of serotypes (4, 6, 11, 19, and 21). Thus, in this patent, the inventors have found that the spa gene is also present in other Erypererotrix species (serotype 18) in addition to the remaining Er serotype. When the amino acid sequences deduced from the base sequences of these genes are examined, there are three types of Spas with low homology of about 60% in Er and other species of the genus Erypererotrix. I found out. In particular, it has been found that Spa possessed by other species of the genus Elyciperotrix (serotype 18) has the broadest protective activity against infection of various serotypes. Therefore, it was shown that the new Spa discovered this time becomes a useful component of a swine erysipelas preventive vaccine, and the present invention has been completed.
J.Syst.Bacteriol., 42, 469-437, 1992 Microb.Pathog., 25, 101-109, 1998 Infect.Immun., 67, 1646-1651, 1999 Infect.Immun., 67, 4376-4382, 1999

  An object of the present invention is to provide a polypeptide exhibiting protective antigen activity against a number of Er serotypes having high safety and excellent efficacy, and a method for producing the same. Another object of the present invention is to use the above polypeptide as a component of a subunit vaccine for the prevention of swine erysipelas infection and to use it as an antigen for antibody measurement and diagnosis against Er.

  For this reason, the present inventors have (a) Er serotype reference strains, particularly serotypes 4, 6, 11, 19 and 21 in which the presence of the spaA gene is not known, and other species of the genus Elysperotrix. Cloning Spa related genes, (b) determining their entire base sequences, and classifying Spa related polypeptides by performing homology searches based on amino acid sequences predicted from the base sequences, (C) The full length of the classified Spa-related gene is incorporated into an expression vector, and the recombinant polypeptide is expressed and purified. Immunize mice and select the polypeptide with the broadest spectrum of protection by cross-protection test. (D) Identify the partial fragment with the highest expression level and excellent yield for the selected Spa polypeptide. , (E) actually immunizing pigs with the obtained polypeptide as a component of the vaccine, attacking with live bacteria of Er to confirm infection / onset protection effect, and (f) using the polypeptide, Research has been conducted with the aim of measuring antibodies in immunized pigs.

  As a result, the inventors synthesized a modified DNA primer based on the base sequence of the spaA gene, and Er serotypes 4, 6, 11, 19, and 21 reference strains for which the presence of spaA was not known. And other serotype 18 strains belonging to species 2 of the genus Erichiperotorix were also confirmed to have Spa-related genes. Thus, the Spa-related genes of all Er serotype reference strains were cloned into vector DNAs and their entire base sequences were determined. As a result, serotypes 1a, 1b, 2, 5, 8, 9, 12, 15, 16, 17 and N carry the spaA gene, serotypes 4, 6, 11, 19 and 21 carry another Spa-related gene (named spaB), and serotype 18 carry another Spa-related gene It was found that there are three types of genes (named spaC). Next, the spaA, spaB, and spaC genes were each expressed in E. coli, and mice were immunized with each Spa polypeptide. On the other hand, when cross-protection tests were conducted by attacking with Er serotypes having different Spas, it was found that SpaC polypeptide exhibited a protective effect against the widest range of serotypes. Next, for the purpose of improving the expression level of SpaC polypeptide in E. coli, a new deletion mutant polypeptide (named SpaCΔC) from which 237 amino acids near the C-terminal were removed was constructed. As a result, the expression level of the SpaCΔC polypeptide in Escherichia coli increased about 10 times compared to the full-length SpaC polypeptide, and the protective activity per protein amount was not different from that of the SpaC polypeptide. From this, it was found that SpaCΔC polypeptide is more suitable as a component of swine erysipelas preventive vaccine, compared to full-length SpaC. Further, since an antibody against Spa polypeptide in the sera of immunized pigs was detected by performing ELISA using the polypeptide as an antigen, SpaCΔC polypeptide is also useful as an antigen for detecting an antibody against Er. It was shown together that the present invention was reached.

That is, the present invention has the following points.
(1) The polypeptide shown in the following (a), (b), (c) or (d).
(A) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) a polypeptide having 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 in the sequence listing, and having an infection, disease protective immune activity against Er,
(C) a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing,
(D) A polypeptide having 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: 3 in the sequence listing, and having infection- and disease-protective immune activity against Er.

(2) DNA encoding the polypeptide according to (1).

(3) The DNA according to (2), which has a nucleotide sequence shown in SEQ ID NOs: 2 and 4 in the sequence listing.

(4) A recombinant expression vector comprising the DNA according to (2).

(5) A transformant transformed with the expression vector according to (4) so as to produce a polypeptide having protective activity against Er infection.

(6) The transformant according to (5) is cultured, the transformant is made to produce the polypeptide according to (1), and the polypeptide is purified, A method for producing a peptide.

(7) A prophylactic vaccine against swine erysipelas comprising the polypeptide according to (1) as a component.

(8) Monoclonal and polyclonal antibodies that recognize the polypeptide according to (1).

(9) Er detection method, antibody comprising using the polypeptide according to (1), the DNA according to (2), the transformant according to (5) or the antibody according to (8) Test method and diagnostic reagent for swine erysipelas.

  According to the invention of claim 1 of the present application, when a pig is immunized, a polypeptide that completely protects against infection and pathogenesis against an attack of an Er toxic strain can be obtained. A pharmaceutical composition that can be used as a vaccine can be provided.

  In addition, according to the invention of claim 2, a gene encoding this polypeptide is obtained, and expressed by the genetic technique of claims 4, 5 and 6, the polypeptide can be produced in a large amount and at a low cost by a simple process. Can be manufactured.

  Further, according to the inventions of claims 8 and 9, antibody detection for obtaining an antibody specific for the polypeptide of claim 1 and using this for diagnosis of swine erysipelas or understanding the immune status of vaccinated pigs Can be implemented.

<1> Polypeptide of the present invention
The polypeptide of the present invention is a polypeptide having the amino acid sequence shown in, for example, SEQ ID NOs: 1 and 3 defined in the above (a), (b), (c) or (d), which induces protective immunity against Er, and variations thereof Provide the body. The SpaC polypeptide shown in SEQ ID NO: 1 is a polypeptide having a molecular weight of 76 kDa, which is specific for other E. cerevisiae species 2 serotype 18. This polypeptide can be obtained by translating it into a polypeptide by various methods based on the DNA shown in SEQ ID NO: 2 encoding the polypeptide.

  The SpaCΔC polypeptide having the amino acid sequence shown in SEQ ID NO: 3 has a molecular weight of 27 kDa, which is a portion of 427 amino acids from the N-terminal of the polypeptide of SEQ ID NO: 1, that is, 237 amino acids deleted from the C-terminal of the SpaC polypeptide. It is a deletion mutant having This polypeptide removes a part of the anchoring region and hydrophobic region to the bacterial surface layer located in the C-terminal region of the SpaC polypeptide, and is limited to only a region related to protective immunity. This SpaCΔC polypeptide has the advantage that its translation efficiency is much higher than that of full-length SpaC when translating a gene into a polypeptide using a transformant such as Escherichia coli. In addition, the ability of the SpaCΔC polypeptide to induce protective immunity per protein amount is equivalent to that of the SpaC polypeptide. This polypeptide can be obtained by translating the DNA shown in SEQ ID NO: 4 encoding the polypeptide by various methods.

  The polypeptide of the present invention has an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequences shown in SEQ ID NOs: 1 and 3, and has an ability to induce protective immunity against Er. The indicated polypeptides are also included. Although there is no limitation on the manner of such mutation, for example, based on the DNA sequence encoding the polypeptide shown in SEQ ID NOs: 1 and 2, artificially mutated DNA can be prepared using techniques such as oligonucleotide site-directed mutagenesis. It can be obtained by preparing and translating it into a polypeptide. In addition, when the spaC gene is cloned from other strains of serotype 18 and translated, polypeptides having SEQ ID NOs: 1 and 3 and several amino acids different from each other may be naturally obtained. Is done. Furthermore, since the SpaCΔC polypeptide shown in SEQ ID NO: 3 is a mutant lacking the C-terminal 237 amino acids based on SpaC, it actually has 1 to 237 amino acids at the C-terminal between SpaC and SpaCΔC. All deletion mutants are also encompassed by the present invention. Expressing the polypeptides encompassed by the present invention separately, the homology with the amino acid sequences of SEQ ID NOs: 1 and 3 should be 70% or more, preferably 80% or more, more preferably 90% or more, and particularly 95% or more. It is.

<2> DNA of the present invention
The DNA of the present invention provides a DNA encoding a polypeptide having the amino acid sequence shown in SEQ ID NOs: 1 and 3 defined in (a), (b), (c) or (d) above and a variant thereof . According to a preferred embodiment, the DNA is DNA having the base sequences shown in SEQ ID NOs: 2 and 4. The DNA of the present invention can be obtained by cloning from the chromosomal DNA, preferably serotype 18, of other species of the genus Eryciperotrix. For example, a genomic library is prepared from the chromosomal DNA of the bacterium, each clone is transformed into a host such as Escherichia coli, and a polypeptide that is encoded is expressed in the host, and a polyclonal or monoclonal antibody against the polypeptide is used. It can be obtained by a method of screening an expression library or a method of labeling DNA containing a part of the nucleotide sequence of SEQ ID NO: 2 or 4 with a radioactive compound, DIG or the like and screening by hybridization using this as a probe. As another method, PCR may be performed using a part of the sequence of SEQ ID NO: 2 or 4 as an oligonucleotide primer, chromosomal DNA of the bacterium as a template, and the amplified DNA fragment cloned into a vector DNA. is there. It is also possible to directly chemically synthesize a sequence based on the nucleotide sequence of SEQ ID NO: 2 or 4 using a DNA synthesizer.

  The DNA defined in claim 2 or 3 has a sequence in which a plurality of base sequences are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2 or 4, and induces protective immunity against Er Also included is a DNA encoding a polypeptide having activity. Although there is no limitation on the method of such mutation, for example, based on the base sequence shown in SEQ ID NO: 2 or 4, a mutant DNA can be artificially prepared using an oligonucleotide site-specific mutation technique or the like. In addition, the spaC gene derived from other strains of the genus Erysiperotic species 2 serotype 18 may have SEQ ID NO: 2 and a plurality of bases substituted. Further, since SEQ ID NO: 4 is a deletion of 714 base pairs at the 3 'end of SEQ ID NO: 2, those having this deletion in the range of 1 to 714 are included in the present invention. Speaking of base sequence homology with SEQ ID NOs: 2 and 4, the mutant should have a homology of 70% or more, preferably 80% or more, more preferably 90% or more, particularly 95% or more. It is. These mutant DNAs can be exemplified as DNA that hybridizes with the DNA of SEQ ID NO: 2 or 4 under a stringent condition such as at 37 ° C. in a hybridization solution containing 50% formaldehyde.

<3> Vector of the present invention, transformant and method for producing polypeptide using transformant
The present invention relates to a recombinant DNA obtained by incorporating the DNA of the present invention into a vector, a transformant obtained by transforming the recombinant DNA, and a culture solution obtained by culturing the transformant A method for producing the polypeptide, comprising recovering the SpaC or SpaCΔC polypeptide from the polypeptide.

  Those capable of autonomous replication or integration into chromosomes in prokaryotic or eukaryotic host cells when transforming the spaC and spaCΔC genes and mutant genes thereof as defined in claims (2) and (3) Can be selected. For example, pUC series (made by Takara Shuzo), pQE series (made by Promega), pBluescriptII series (made by Stratagene), pET series (made by Novagen), pHY300PLK (made by Takara Shuzo), etc. for prokaryotic cells PSG5 (manufactured by Stratagene), pBPV (manufactured by Pharmacia), pcDM8 (manufactured by Funakoshi), pREP4 (manufactured by Invitrogen), etc. for animal cells, pBlueBacIII (manufactured by Invitrogen), etc. Examples of plant cells include, but are not limited to, Ti plasmids and tobacco mosaic virus vectors. Of course, it is also possible to arbitrarily modify commercially available or literature-described vectors as necessary.

  A transformant can be obtained by introducing the DNA of the present invention into an appropriate host and transforming it. Host cells include, for example, microorganisms such as Escherichia and Bacillus as prokaryotes, yeasts such as Saccharomyces, Schizosaccharomyces and Pichia as eukaryotes, and human fetal kidney cells Chinese hamster ovary (CHO) cells. Examples include mammalian cells, baculovirus-sensitive insect cells, insect bodies, etc. Transformation methods are not limited to the following, but include the calcium chloride method, the calcium phosphate method, the lithium acetate method, the electroporation method, and the protoplast method. , Spheroplast method, lipofection method, Agrobacterium method and the like can be used.

  The transformed host cell is cultured, the target DNA incorporated in the expression vector is expressed, and SpaC, SpaCΔC and its mutant polypeptide can be separated and purified using known methods for protein purification. Usually, the host cells are destroyed by ultrasonic treatment, homogenizer treatment, high pressure compression treatment, etc. to obtain an extract, and from this extract, solvent extraction, salting out, desalting, organic solvent precipitation, ultrafiltration, ion exchange, By combining methods such as hydrophobic interaction, HPLC, gel filtration and affinity chromatography, electrophoresis, and isoelectric focusing, the separated and purified polypeptide can be obtained.

<4> Swine erysipelas preventive vaccine comprising the polypeptide of the present invention as a component, monoclonal and polyclonal antibodies, and bacteria detection method and antibody test method using the antibody The polypeptide obtained by the present invention is a subject animal for swine erysipelas. By immunizing pigs and laboratory animals, antibodies against the polypeptide are induced in the blood of the animals, and the action of the antibodies protects the animals from infection and disease against attack by strongly toxic bacteria of Er. The Due to such effects, the polypeptide can be used as a vaccine against swine erysipelas. In addition, since the polypeptide has high immunogenicity as described above, it is possible to produce antibodies against various animals. The antibody produced in the serum is derived from the spleen cells of the immunized mouse as a polyclonal antibody. The antibodies produced by the hybridomas obtained above can be used as monoclonal antibodies. Since these polyclonal and monoclonal antibodies react specifically with Er, by applying various antigen-antibody reactions, detection of Er from infected animals, detection of antibodies against Er produced in infected and immunized animals, and It can be applied to quantification.

  A single dose of the polypeptide when used in a vaccine preparation is usually 1 μg protein or more, preferably 10 μg protein or more, more preferably 100 μg protein or more, particularly 100 μg to 1000 μg protein. When a polypeptide is used as a vaccine, it is preferable to add an immunostimulant (adjuvant) in order to further enhance the antibody response. As an adjuvant, aluminum hydroxide gel, aluminum phosphate gel, a mixture of vegetable or mineral oils and surfactants, etc. can be used, and they are formulated in an immunostimulatory amount according to the characteristics of each adjuvant. The

  In addition to the polypeptide of the present invention, an antigenic substance may be mixed in the vaccine preparation of the present invention. For example, porcine-derived pathogenic bacteria or pathogenic virus-derived antigens are preferred, and more specifically, Bordetella bronchiseptica, Pasteurella multocida, Mycoplasma hyopeumoniae, Actinobacillus・ Bronlopneumoniae (Actinobacillus pleuropneumoniae), Haemophilus parasuis, Esherichia coli, Salmonella choleraesuis, Streptococcus suis, Porcine reproductive and respiratory syndrome virus (Porcine virus) ), Japanese encephalitis virus, porcine parvovirus, porcine circovirus, swine influenza virus, Aujeszky's disease Illus (Aujesky ’s disease virus) and the like can be mentioned. A pathogen-derived antigen is a recombinant protein expressed in other organisms such as Escherichia coli or baculovirus based on the gene of the microorganism or virus itself, or extracted or purified from the microorganism or virus. Also good.

  The evaluation of the vaccine effect can be performed by an immunization / attack test using experimental animals such as mice or pigs which are target animals. For example, the following may be performed. The polypeptide is mixed with an adjuvant and injected twice into the neck muscles of pigs in 1 mL increments every 3-4 weeks. The control group is non-immunized. Two weeks after the second injection, the patient is challenged by injecting viable Er toxic bacteria into the skin and observed for about one week after the challenge. When comparing the number of deaths in the vaccine injection group and the control group and the number of clinical symptoms of swine erysipelas, the vaccine effect can be determined by the fact that the vaccine injection group is significantly less than the control group. When evaluating using a mouse, a mixture of the polypeptide and an adjuvant is injected subcutaneously twice with 0.2 mL each at an interval of 3 weeks. The control group is non-immunized. Two weeks after the second injection, the rats are challenged by injecting live virulent Er bacteria into the skin of each mouse and observed for about 2 weeks after the challenge. Since the number of deaths in the vaccine injection group is significantly smaller than that in the control group, the effect of the vaccine can be determined.

  A polyclonal antibody against the polypeptide of the present invention can be obtained, for example, as follows. The polypeptide is mixed with adjuvant and immunized 3-5 times subcutaneously at the back of the rabbit at 2-4 week intervals. Whole blood is collected 2-3 weeks after the final immunization to obtain immune serum. From this serum, a polyclonal antibody is obtained by collecting an immunoglobulin fraction using an ammonium sulfate fractionation method or a commercially available antibody purification kit. A monoclonal antibody against the polypeptide of the present invention can be obtained, for example, as follows. Spleen cells are collected from the mouse immunized as described above, and fused cells (hybridoma) with mouse myeloma cells are prepared according to a conventional method (MONOCLONAL ANTIBODYTECHNOLOGY, Elsevier Science Publishers, Amsterdam, 1984). Among these, after screening for a hybridoma confirmed to produce an antibody against the polypeptide, and further cloning the cell line, the cell line was cultured or the ascites of nude mice transplanted with the cell line. A monoclonal antibody can be obtained by collecting the immunoglobulin fraction by the method described in 1. above.

  Polyclonal and monoclonal antibodies against the polypeptide of the present invention can be applied to the detection and quantification of antibodies present in the serum of swine erysipelas-affected pigs and swine erysipelas-immunized pigs. The antibody measurement result is an important index in diagnosing swine erysipelas or grasping the immune state by swine erysipelas vaccine injection. Moreover, it can be applied as a reagent for detection of Er from organs and joints of swine erysipelas-affected pigs. By detecting Er, it is possible to diagnose whether the animal suffers from swine erysipelas.

  For example, an enzyme linked immunosorbent assay (ELISA) using an antibody against the polypeptide can be applied to the measurement of antibodies in the serum of swine erysipelas-affected pigs as follows. Rabbit polyclonal antibody diluted with carbonate / bicarbonate buffer pH 9.6 or the like is dispensed onto a commercially available ELISA plate and adsorbed overnight at 4 ° C. After blocking with a 1% gelatin solution or the like, the polypeptide of the present invention diluted with a buffer solution is added and reacted at 37 ° C. for about 30 minutes. Next, test pig serum is added, and after reaction at 37 ° C. for about 30 minutes, anti-pig IgG enzyme-labeled antibody is added and allowed to react at 37 ° C. for 15 minutes. After washing, add a substrate / coloring agent mixture (0.04% orthophenylenediamine, 0.02% hydrogen peroxide-containing citric acid / phosphate buffer pH 5.0, etc.) and react at room temperature for about 20 minutes. . Stop the reaction by adding a stop solution (4N sulfuric acid, etc.), and measure the absorbance value at 492 nm. The degree of absorbance indicates the antibody level against the polypeptide contained in the test serum. If this value is high in non-vaccinated pigs, it can be diagnosed that there was an Er infection. On the other hand, in the vaccine-injected pigs, the degree of acquisition of protective immunity induced by the vaccine injection can be known by examining the level of this value.

  On the other hand, Er can be detected by applying the same ELISA method. As described above, a monoclonal antibody against the polypeptide of the present invention diluted with a carbonate / bicarbonate buffer or the like is adsorbed on an ELISA plate. After blocking, the blood of the pig suspected of being infected with Er, the joint fluid, the emulsion of organs, lymph nodes, etc. is diluted with a buffer solution, added to the plate, and reacted at 37 ° C. for about 30 minutes. After washing, a rabbit polyclonal antibody against the polypeptide of the present invention is added and reacted at 37 ° C. for about 30 minutes. After washing, a commercially available anti-rabbit IgG enzyme-labeled antibody is added thereto and further reacted at 37 ° C. for about 15 minutes. After washing, a substrate / coloring agent mixed solution is added and reacted at room temperature for about 20 minutes. Then, a stop solution is added to stop the reaction, and an absorbance value at 492 nm is measured. When Er is present in the sample, it shows a high absorbance value, so that the presence of Er in the sample can be detected more quickly than in the culture method, and the test pig suffers from swine erysipelas. It is possible to quickly diagnose whether or not it exists.

  In addition to those exemplified above, antibody measurement and Er detection can be carried out using the polypeptide of the present invention by a well-known antigen-antibody reaction, such as Western blotting, latex agglutination, fluorescent antibody method and radioimmunoassay. it can.

Examples Hereinafter, the present invention will be described more specifically with reference to examples.

Cloning of Er spaA and its related genes Each serotype known to have the Er spaA gene, and Er serotype and other erythiperotics for which no spaA gene has been previously detected An attempt was made to clone spaA and its related genes from the genus species.

  Each serotype reference strain of Er was statically cultured at 37 ° C. for 18 hours in 0.1% Tween 80, 1% bovine serum-added tryptose phosphate broth. The cells recovered by centrifugation were suspended in sterilized distilled water, heat-treated at 100 ° C. for 5 minutes, and then centrifuged to recover the supernatant. Using the chromosomal DNA contained in this supernatant as a template, PCR was performed using two types of oligonucleotide primers Er60K-1F (FIG. 1, SEQ ID NO: 7) and Er60K-2RH (FIG. 1, SEQ ID NO: 8). As a result, in each of the serotype strains of Er serotypes 1a, 1b, 2, 5, 8, 12, 15, 16, 17 and N, which are known to have spaA, about 1.9 A kb gene fragment was amplified (FIG. 2). Next, serotypes 4, 6, 11, 19 which are serotypes of Er and other species of the genus Elyciperotrix, for which the presence of spaA gene was not known and amplification products were not obtained by the above primers , 21 and 18, two new oligonucleotide primers Er70-1F (FIG. 1, SEQ ID NO: 9) and Er70-2R (FIG. 1, SEQ ID NO: 10), which are different in sequence from the above primers, were synthesized. PCR was performed again. As a result, a DNA amplification product of about 1.9 kb from Er serotypes 4, 6, 11, 19 and 21 and a DNA amplification product of about 2 kb from serotype 18 which is another species of the genus Eryciperotrix. (FIG. 2). The PCR products amplified from these serotypes were purified by phenol / chloroform extraction and ethanol precipitation, and then cloned into vector plasmids (pGEM-T Easy, manufactured by Promega) according to conventional methods. This was transformed into Escherichia coli XL-1 Blue (manufactured by Stratagene) by electroporation, and cultured on an LB agar medium containing 50 μg titer / mL ampicillin at 37 ° C. for 18 hours. The formed colonies are inoculated into ampicillin-containing LB liquid medium and cultured with shaking at 37 ° C. for 18 hours, and each plasmid DNA is obtained from the resulting bacterial solution using a commercially available kit (Qiagen Plasmid Kit, manufactured by Qiagen). Was extracted and purified.

Comparison of nucleotide sequences of spaA and related genes and comparison of deduced amino acid sequences of polypeptides encoded thereby SpaA and related genes derived from each serotype bacterium cloned into a plasmid are commercially available kits (BigDye Terminator Cycle Sequencing Ready Reaction After the dideoxy sequencing reaction using Kit, Applied Biosystem (ABI), the entire base sequence was determined using a fully automatic base sequencer (A310, manufactured by ABI). The open reading frame (ORF) was searched based on the obtained base sequence, and the amino acid sequence of SpaA derived from each serotype of Er encoded by this ORF and its related polypeptide was estimated.

  Table 1 shows the results of a homology search on the deduced amino acid sequences of SpaA-related polypeptides derived from Er serotypes that had been amplified by PCR and other species of the genus Erysiperotics. Indicated. When polypeptides having a homology of 95% or more were grouped into one group, each polypeptide was clearly divided into three groups. Serotype 1a, 1b, 2, 5, 8, 12, 15, 16, 17 and N polypeptides belonged to group 1 which showed 95% or more homology with SpaA. On the other hand, polypeptides derived from serotypes 4, 6, 11, 19 and 21 belonged to group 2 showing homology of only about 60% with SpaA, but 95% or more of each other. Furthermore, a polypeptide derived from serotype 18 (other species of genus Elyciperotrix) belonged to group 3, which showed only about 60% homology with both the SpaA and group 2 polypeptides. As described above, it has been clarified that Er and other species of the genus Eryciperotrix include at least three clearly different polypeptides including SpaA as a cell surface protective antigen. Therefore, since the conventionally reported serotype 1 and type 2 polypeptides are named SpaA, the polypeptides of Er serotypes 4, 6, 11, 19 and 21 discovered this time are SpaB and others. The polypeptide possessed by the serotype 18 of the genus Eryciperotrix sp. Was newly named SpaC.

  When three types of Spa polypeptides were compared for amino acid homology by intramolecular functional domain, the signal region containing 29 amino acids from the N-terminus was identical in sequence to SpaA, SpaB and SpaC (FIGS. 3, 4). 5 and 6). In the repeat unit region around the C-terminal, high homology was observed between the polypeptides, but the number of repeats was 9 for SpaA and SpaB, while 10 for SpaC. The intermediate domain considered to be involved in protective antigenicity showed the lowest homology between the polypeptides, and was about 40-50%. Therefore, when each Spa polypeptide was used as a component of a vaccine, the possibility of showing different protective antigenicity was suggested.

  From the above results, it has been clarified for the first time that Er and other types of E. peritrix have three types of cell surface protective antigens. In the following, SpaA is a serotype 1a Fujisawa strain, SpaB is a serotype 6 Dolphin E-1 strain, and SpaC is a polypeptide derived from serotype 18 715 strain belonging to other species of the genus Eryciperotrix. Used for subsequent tests.

Expression and purification each Spa polypeptide of the Spa polypeptide, to express in recombinant E. coli as a fusion protein with a histidine hexamer, was cloned in the following manner each gene into vectors pQE series.

  For the spaA and spaB genes, each plasmid DNA was double-digested with BamHI and HindIII to excise a DNA fragment of about 1.9 kb, and ligated to the pQE9 vector cut with the same restriction enzymes. The spaC gene was double-digested with BamHI and SalI to excise a DNA fragment of about 2 kb, and ligated to the pQE30 vector cut with the same restriction enzyme. Each ligated body was transformed into Escherichia coli XL-1 Blue by electroporation, and cultured on an LB agar medium containing ampicillin to form colonies.

  Colonies formed on the plate were selected, inoculated into 250 mL of LB liquid medium containing ampicillin, and cultured with shaking at 37 ° C. for 18 hours. The bacterial cells are collected from this culture solution by centrifugation, resuspended in 250 mL of LB medium containing 1 mM isopropylthiogalactoside (IPTG), and further cultured by shaking at 37 ° C. for 4 hours. Expressed. Escherichia coli expressing each polypeptide was collected by centrifugation, and the cells were disrupted by ultrasonic treatment several times for 20 seconds at level 5 using an ultrasonic disruption device (Sonic Power, manufactured by Branson). This was centrifuged at 10,000 × g for 20 minutes, and each precipitated Spa polypeptide was suspended in 3.5 M guanidine buffer (pH 7.4) and solubilized. Each solubilized Spa polypeptide was purified by nickel resin affinity column chromatography (manufactured by Chiadien) according to the attached instruction manual.

The ability of SpaA, B and C polypeptides to induce cross-protective immunity
Use 160 5-week-old ddY mice, divided into 4 groups of 40 mice each, and 40 μg protein / mouse of SpaA, SpaB, and SpaC, respectively, equivalent to commercially available oil-in-water adjuvant (manufactured by Sepic) They were mixed and immunized by subcutaneous injection twice at 3-week intervals. The remaining 40 animals served as non-injection controls. Two weeks after the second injection, each group was further divided into 4 groups of 10 animals, and 0.1 ml of live bacteria of Er serotypes 1a, 2, 6 and 18 were subcutaneously inoculated and challenged. The amount of attack bacteria was 2.9 × 10 ² , 3.8 × 10 ² , 4.1 × 10 ⁴ and 6.9 × 10 ² CFU in this order. Observation was made for 2 weeks after the attack, and the protective effect was determined by life and death.

  The non-injected control group all died against any serotype challenge (Table 2). High protection was observed in both the SpaA and SpaC immunization groups against the attack of serotype 1a and type 2 bacteria carrying SpaA, whereas in the SpaB immunization group, 9 animals against Five animals died against the mold attack and no protective effect was observed. Similarly, against the serotype 6 challenge carrying SpaB, the SpaB and SpaC immunized groups were protected, but in the SpaA immunized group 5 animals died and the degree of protection was insufficient. High protection was observed in the SpaC immunity group against the attack of serotype 18 carrying SpaC, but 3 and 4 animals died in the SpaA and SpaB immunization groups, respectively, and the degree of protection was moderate. It was. From the above results, it was found that the SpaC polypeptide is the one that exhibits the highest cross-protective antigenicity against the attack of each serotype among three types of Spa.

Production of SpaCΔC polypeptide and its expression in full-length SpaC polypeptide in immunogenic Escherichia coli was low. Thus, when the SpaC molecule is viewed by functional region (domain), repeat regions existing near the C-terminal and hydrophobic regions rich in proline and glycine are examples of other Gram-positive bacteria such as Streptococcus and Clostridium (Pancholi, V. and Fischetti, VA, J. Bacteriol., 170, 2618-2624, 1988; Yother, J. and Briles, DE, J. Bacteriol., 174, 601-609, 1992) It seemed to be related to the binding to the surface of the cells. Therefore, since this region was considered not to be directly involved in protective antigenicity, 714 bp was deleted from the 3 ′ end of the spaC gene for the purpose of producing a polypeptide with 237 amino acids removed from the C terminus (hereinafter referred to as SpaCΔC). The constructed DNA fragment was constructed. In order to amplify the fragment, two types of oligonucleotide primers, Er70-1F (FIG. 1, SEQ ID NO: 9) and Er70K18-2R (FIG. 1, SEQ ID NO: 11) were synthesized, and PCR was performed using the spaC gene as a template. . The amplified DNA fragment of about 1.3 kb was cloned into the pGEM-Easy vector in the same manner as in Example 2, and the nucleotide sequence was determined to confirm that the predetermined DNA fragment was inserted. Subsequently, in order to express the SpaCΔC polypeptide encoded by this gene fragment in Escherichia coli as a fusion protein with histidine hexamer, the gene fragment was excised by cutting with restriction enzymes BamHI and SalI in the same manner as in Example 2. It was cloned into a pQE30 expression vector cut with the same enzyme. In Escherichia coli transformed with this plasmid, expression of a large amount of polypeptide was observed at a position of about 49 kDa, which is the molecular weight expected from the sequence of SpaCΔC polypeptide (FIG. 7). On the other hand, in E. coli transformed with a plasmid containing the full length spaC gene, a thin band was observed in the vicinity of 76 kDa corresponding to the molecular weight of SpaC and in the vicinity of 50 to 40 kDa which seems to be a degradation product thereof. The SpaCΔC polypeptide was extracted by crushing the expressed E. coli and purified in the same manner as in Example 2 using nickel resin affinity column chromatography.

An attempt was made to confirm whether this SpaCΔC polypeptide is immunogenic, similar to the original SpaC molecule, and whether cross-protection against the attack of multiple serotypes has been lost when immunized. Ninety five-week-old ddY mice were used, 30 full length SpaC polypeptides were prepared, the other 30 were SpaCΔC polypeptides, each with an amount of 40 μg protein per mouse, and an adjuvant. Two subcutaneous injections were made at 3 week intervals. The remaining 30 animals served as a non-immunized control group. Two weeks after the second injection, each group was further divided into 3 groups of 10 animals, and each 0.1 ml of Er serotype 1a, 6 or 18 live bacteria was subcutaneously inoculated and challenged. The amount of attacking bacteria was 3.3 × 10 ² CFU (type 1a), 4.6 × 10 ⁴ CFU (type 6), and 7.1 × 10 ² CFU (type 18), respectively. Observation was made for 2 weeks after the attack, and the protective effect was determined by life and death.

  In mice immunized with the full-length SpaC polypeptide, 7 out of 10 mice survived by type 1 challenge, 7 by type 6 challenge, and 8 by type 18 challenge (Table 3). On the other hand, in mice immunized with SpaCΔC polypeptide, 10 mice survived type 1 challenge, 9 survived type 6 challenge, and 9 survived type 18 challenge.

  From the above results, the SpaCΔC polypeptide from which 237 amino acids present at the C-terminal were removed had an immunogenicity equivalent to or higher than that of the full-length SpaC polypeptide, and the cross-protective ability was ensured. This is because Shimoji et al. (Infect. Immun, 67: 1646-1651, 1999) that protective antigenicity exists in the Spa polypeptide from the N-terminal to the middle part and not in the repeat region near the C-terminal. Was consistent with the report. Since the SpaCΔC polypeptide has an expression efficiency of 10 times or more in Escherichia coli as compared with the full-length SpaC, it was considered to be extremely useful as a component of a swine erysipelas preventive vaccine.

Immunity test in pigs of SpaCΔC polypeptide
The immunogenicity of SpaCΔC polypeptide was confirmed using pigs, which were actual target animals.

Nine 4 week old SPF pigs were used for the test. Each group is divided into 3 groups, and SpaCΔC polypeptide is prepared to have a protein amount of 200 μg per head in the first group. 11 (Sepic) and two injections at 3-week intervals. Group 2 contains the same amount of SpaCΔC polypeptide as Group 1, commercially available swine adjuvant No. The same injection was carried out together with 13 (manufactured by Sepic). Group 3 was a non-injection control. Each immunized pig was challenged intradermally with Er Fujisawa strain (serotype 1a) 1.0 × 10 ⁶ CFU together with the control group 2 weeks after the final immunization. One week after the challenge, clinical observations were made, necropsied at the end of the study, and each organ was observed macroscopically and bacteria were isolated. In addition, blood was collected from each group of pigs before immunization and at the time of challenge, and antibodies against SpaCΔC polypeptide produced in the serum were examined by ELISA described below.

  To each well of a commercially available 96-well microplate for ELISA, a rabbit polyclonal antibody against SpaCΔC polypeptide diluted 10,000-fold with carbonate bicarbonate buffer pH 9.6 was added and allowed to stand overnight at 4 ° C. for adsorption. After washing with physiological saline supplemented with 0.05% Tween 80, 1% gelatin solution was added, and the mixture was allowed to stand at room temperature for 1 hour or longer to block. After washing, 0.25 μg protein amount of SpaCΔC polypeptide was added and reacted at 37 ° C. for 30 minutes. Next, after washing, porcine serum diluted 100-fold with PBS (−) containing 0.05% Tween 80-1% gelatin was added and reacted at 37 ° C. for about 30 minutes. After washing, an anti-pig IgG peroxidase-labeled antibody (manufactured by Rockland) diluted 10,000 times was added and reacted at 37 ° C. for 15 minutes. After washing, a substrate / coloring agent mixture (0.04% orthophenylenediamine, 0.02% citrate / phosphate buffer solution containing hydrogen peroxide, pH 5.0) was added and reacted at 30 ° C. for 20 minutes. Sulfuric acid was added to stop color development. Absorbance (OD) at 492 nm was measured for each well, and E value [(OD value of test serum−OD value of indicator-negative serum) ÷ (OD value of indicator-positive serum−OD value of indicator-negative serum)] was calculated. . An E value of 0.1 or higher was determined as positive.

  None of the vaccine group pigs immunized with SpaCΔC polypeptide using two types of commercially available pig adjuvants had side reactions such as fever and depression associated with injection, and redness, induration, etc. There was no abnormality, and it was confirmed that SpaCΔC polypeptide can be safely used as a component of a vaccine for swine.

  After challenge with viable Er against each group, the control group exhibited severe clinical symptoms and all animals died (Table 4). Aggressive bacteria were isolated in pure culture form from each organ of these dead pigs. On the other hand, both vaccine groups immunized with SpaCΔC polypeptide using two types of adjuvants showed no clinical symptoms and no dead pigs. At autopsy, no challenge bacteria were recovered from any pig organ.

  In the antibody test, no antibody against SpaCΔC polypeptide was detected in all pigs before the start of the test. Two weeks after the second injection (at the time of challenge), antibodies were seroconverted in all pigs of both vaccine groups immunized with SpaCΔC polypeptide. On the other hand, all three of the control group passed negative.

  From the above results, it was found that the SpaCΔC polypeptide can produce antibodies in pigs, and can provide a high level of protection against live Er attack by immunization, using any commercially available pig adjuvant. did. From this, it was demonstrated that SpaCΔC polypeptide is useful as a component of a swine erysipelas preventive vaccine.

  In addition, it was possible to detect antibodies raised by vaccine injection using rabbit polyclonal antibody against SpaCΔC polypeptide by ELISA, and the antibody test results by this ELISA and the protective effect by the attack test were in good agreement. Therefore, the antibody level against Er can be known by carrying out antibody measurement using SpaCΔC polypeptide as an antigen.

Immunity test in pigs using SpaCΔC polypeptide prepared in a simple process In Example 6, a highly purified SpaCΔC polypeptide was used as an immunogen using nickel resin affinity column chromatography, and had anti-infection activity. Was confirmed. However, in order to actually use it as an antigen for a vaccine, it is necessary to establish a method for obtaining a polypeptide in a large amount and at a low cost although it is somewhat inferior in purity. Therefore, it was examined whether SpaCΔC polypeptide prepared by a simple process as described below retains immunogenicity in pigs.

  The Escherichia coli expressing SpaCΔC polypeptide as described in Example 3 was collected by centrifugation from the culture medium, and it was collected several times for 20 seconds at level 5 using an ultrasonic crusher (Sonic Power, manufactured by Branson). The microbial cells were crushed by sonication. This was centrifuged at 10,000 × g for 20 minutes, and SpaCΔC polypeptide recovered in the precipitate was suspended in 3.5 M guanidine buffer (pH 7.4) and solubilized. This was centrifuged at 15,000 rpm for 5 minutes at room temperature to remove insoluble precipitates, and the resulting supernatant was used as a SpaCΔC polypeptide solution, which was used for vaccine preparation.

Six SPF pigs of about 4 weeks of age were used, 3 were immunized and the remaining 3 were non-injected control. For the swine in the immunized group, the SpaCΔC polypeptide prepared as described above was prepared so that the amount of protein was 100 μg per head. Two intramuscular injections were made at 13-week intervals with 13 adjuvants. The remaining 3 animals served as non-injection controls. Two weeks after the second injection, Er Fujisawa strain (serotype 1a) 1.2 × 10 ⁶ CFU was inoculated intradermally and challenged. One week after the challenge, clinical observations were made, necropsied at the end of the study, and Er was isolated from each organ.

  After challenge, the control group exhibited severe clinical symptoms and all animals died (Table 5). Er was isolated from each organ of the dead pig in a pure culture. On the other hand, none of the pigs immunized with the SpaCΔC polypeptide prepared by a simple process showed clinical symptoms, and Er was not isolated from the organs.

  From the above results, in the SpaCΔC polypeptide obtained by a simple process capable of preparing a polypeptide in large quantities and at low cost, as in the case of highly purified, the attack of the Er toxic strain by immunizing pigs On the other hand, it was confirmed to have an activity capable of completely protecting against infection and disease. Therefore, it was considered that the fact that SpaCΔC polypeptide having sufficient protective antigenicity was obtained by such a relatively simple process was very useful in actual vaccine production.

  According to the invention of claim 1 of the present application, when a pig is immunized, a polypeptide that completely protects against infection and pathogenesis against an attack of an Er toxic strain can be obtained. Since the pharmaceutical composition which can be used as a vaccine can be provided, this invention is useful.

  In addition, according to the invention of claim 2, a gene encoding this polypeptide is obtained, and expressed by the genetic technique of claims 4, 5 and 6, the polypeptide can be produced in a large amount and at a low cost by a simple process. Therefore, the present invention is useful.

  Further, according to the inventions of claims 8 and 9, antibody detection for obtaining an antibody specific for the polypeptide of claim 1 and using this for diagnosis of swine erysipelas or understanding the immune status of vaccinated pigs Therefore, the present invention is useful.

Primers for PCR cloning of SpaA, SpaB, SpaC and SpaCΔC genes in Er serotypes. Detection of SpaA, SpaB, and SpaC genes in Er serotypes by PCR (M is molecular weight marker: 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1 from the top) And 0.5 kb) Comparison of molecular structure of each Spa polypeptide. Comparison of amino acid sequences of SpaA (serotype 1a) and SpaB (serotype 6) polypeptides. Comparison of amino acid sequences of SpaA (serotype 1a) and SpaC (serotype 18) polypeptides. Comparison of amino acid sequences of SpaB (serotype 6) and SpaC (serotype 18) polypeptides. Electrophoresis photograph of expression in Escherichia coli of full length SpaC and C-terminal region deletion fragment.

Claims (9)

The polypeptide shown in the following (a), (b), (c) or (d).
(A) a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) a polymorphism having 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 in the sequence listing, and having an immunoprotective activity against infection and disease prevention against sweptococcus pneumoniae peptide,
(C) a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing,
(D) a polymorphism having 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: 3 in the sequence listing, and having an immunoprotective activity against infection and disease prevention against sweptococcus pneumoniae peptide.   DNA encoding the polypeptide of claim 1.   The DNA according to claim 2, which has a nucleotide sequence shown in SEQ ID NOs: 2 and 4 in the sequence listing.   A recombinant expression vector comprising the DNA according to claim 2.   A transformant, which is transformed with the expression vector according to claim 4 so as to produce a polypeptide having an immunoprotective activity against infection with swine erysipelas.   The transformant according to claim 5 is cultured, the transformant is produced with the polypeptide according to claim 1, and the polypeptide is purified. Method.   A prophylactic vaccine against swine erysipelas comprising the polypeptide according to claim 1 as a component.   Monoclonal and polyclonal antibodies that recognize the polypeptide of claim 1.   Use of the polypeptide according to claim 1, the DNA according to claim 2, the transformant according to claim 5, or the antibody according to claim 8; Methods and diagnostic reagents for swine erysipelas.