JP2012034652A - Cytokine-expressing erysipelothrix rhusiopathiae and delivery system for cytokine using erysipelothrix rhusiopathiae - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Erysipelothrix rhusiopathiae which can express immunoenhancing effect on non-human animals by safely supplying a cytokine to non-human animal bodies, methods for preventing and/or treating diseases of non-human animals, a system for safely delivering a cytokine to non-human animal bodies, a new vaccine adjuvant, and a vaccine preparation for non-human animals.SOLUTION: The Erysipelothrix rhusiopathiae is a transformant of attenuated strain of Erysipelothrix rhusiopathiae, wherein the Erysipelothrix rhusiopathiae has a recombinant DNA coupled with a gene encoding a cytokine and expresses the cytokine. The cytokine is one or more kinds selected from the group consisting of an interleukin, an interferon, a hemopoietic factor, a cell growth factor and a cytotoxic factor. The Erysipelothrix rhusiopathiae is also suitably used for methods for preventing and/or treating diseases of non-human animals, a system for safely delivering a cytokine to non-human animal bodies, a new vaccine adjuvant, and a vaccine preparation for non-human animals.

Description

  The present invention relates to swine erysipelas expressing cytokines and a system for delivering cytokines into non-human animals.

  Currently, when raising livestock animals such as pigs, cows, chickens, etc., it is generally practiced to administer vaccines for health management, but adjuvants are used to activate the immunity of livestock animals. Has been. However, since adjuvants are inflammation-inducing substances, they can sometimes cause side effects and residual problems in animals used, and the cost is high. Therefore, there is a need for substances that replace adjuvants. As such an adjuvant substitute, cytokines produced by the animals in the body (hereinafter, cytokines derived from animals other than humans are also referred to as animal cytokines) have attracted attention.

  Animal cytokines are produced by cells of the immune system in the animal's body, but need to be manufactured in large quantities to be used like adjuvant substitutes. Several genetic engineering production methods have already been reported as methods for producing such animal cytokines. For example, the present inventors have developed a production method of active interleukin-18 characterized by using porcine interleukin 18 as an cytokine and insect cells or insects (Patent Document 1). In addition, a porcine bone marrow cell-derived cell line that produces porcine interleukin 8 under non-stimulation and a method for producing porcine interleukin 8 using this cell line are known (Patent Document 2). In addition to swine, bovine interleukin can be obtained by culturing Brevibacillus chosinensis transformed with a plasmid incorporating a DNA fragment having a base sequence encoding the amino acid sequence of a protein having bovine interleukin 1β activity. There is also known a method for producing bovine interleukin 1β characterized by producing and accumulating 1β in a culture and collecting it (Patent Document 3).

  In any of the above-mentioned genetic engineering techniques, since a desired cytokine is secreted into the cell culture medium, it is necessary to purify it for use as an adjuvant substitute. However, when such purification is performed, there is a problem that the manufacturing cost increases.

In addition, although several methods for producing animal cytokines are known as described above, to date, only one product that actually uses animal cytokines is limited. This is because it is easily affected by digestive enzymes when animal cytokines are administered orally, and when administered by injection, side effects such as fever are likely to occur because large amounts of animal cytokines are administered at once. This is because there are technical issues and there is still room for further study.
Furthermore, a technique for efficiently and safely delivering animal cytokines into an animal body to express a desired effect has not been established.

Japanese Patent No. 3541216 JP 2004-129660 A JP 2003-135060 A

  The present invention has been made in view of the above circumstances, and it is possible to safely supply cytokines into the body of a non-human animal so that the non-human animal can exert an immunopotentiating action. It is an object of the present invention to provide a method for preventing and / or treating a disease and a system for safely delivering a cytokine into a non-human animal body. Another object of the present invention is to provide a novel vaccine adjuvant and a vaccine preparation for non-human animals.

  As a result of diligent research to solve the above problems, the present inventors have focused on attenuated swine erysipelas out of various host cell candidates so that swine cytokines can be expressed as one of animal cytokines. When the pig erysipelas were transformed and administered to pigs, the present inventors found the fact that an immunopotentiating action derived from swine cytokines was observed and completed the present invention.

That is, the gist of the present invention is as follows.
(1) A porcine venom that is a transformant of an attenuated strain of porcine erythematosus, having a recombinant DNA combined with a gene encoding a cytokine, and expressing the cytokine,
(2) The swine erysipelas according to (1) above, wherein the cytokine is one or more selected from the group consisting of interleukins, interferons, hematopoietic factors, cell growth factors and cytotoxic factors,
(3) The swine venom strain described in the above (1) or (2), wherein the porcine erysipelas attenuated strain serving as the host is the porcine erysomycetes koganei strain 65-0.15 or the capsular-deficient swine erysipelas attenuated YS-1 strain,
(4) A method for preventing and / or treating a disease in a non-human animal, comprising a step of administering the swine erysipelas according to any one of (1) to (3) to a non-human animal,
(5) A cytokine delivery system into a non-human animal, wherein the swine erysipelas described in any of (1) to (3) above is administered to a non-human animal,
(6) A method of using the swine erysipelas according to any one of (1) to (3) as a vaccine adjuvant in a non-human animal,
(7) The present invention relates to a vaccine preparation for non-human animals containing swine erysipelas according to any one of (1) to (3).

The swine venom of the present invention can be administered to a non-human animal and can continuously supply a desired cytokine in an amount that affects immunity while the fungus can survive in the body of the non-human animal. Furthermore, the use of viable bacteria can affect the entire network of various cytokines induced by pathogen infection.
Therefore, prevention or treatment of diseases of non-human animals can be easily performed by using the swine venom of the present invention.
Moreover, since the swine venom of the present invention can enhance the immunity of non-human animals, the combined use of other vaccine preparations makes it easier to obtain the preventive or therapeutic effect in non-human animals.

FIG. 1 is a schematic diagram showing the procedure for producing swine erysipelas YS-1 / IL18 strain in Example 1. FIG. 2 is detected in the culture supernatant and centrifuge supernatant of the capsular-deficient swine erysipelas attenuated strain YS-1 (YS-1 / IL18 strain) into which poIL-18 gene was introduced in Example 1. 2 is a graph showing the content of poIL-18. The control is YS-1 strain (parent strain). FIG. 3 is detected in the culture supernatant and the disrupted cell supernatant of Koganei strain 65-0.15 (KO / IL18 strain) in which the poIL-18 gene was introduced in Example 2. It is a graph which shows content of poIL-18. The control is the KO strain (parent strain). FIG. 4 is a graph showing that in Example 3, mouse splenocytes significantly produced IFN-γ by direct stimulation of the culture supernatant of YS-1 / IL18 strain. The control is YS-1 strain (parent strain). FIG. 5 is a graph showing that in Example 3, the spleen cells of mice collected on day 5 after the YS-1 / IL18 strain was administered to mice significantly produced IFN-γ by stimulation with concanavalin A. It is. The control is YS-1 strain (parent strain). FIG. 6 is a graph showing that, in Example 4, peritoneal macrophages of mice administered with the YS-1 / IL18 strain have enhanced phagocytic ability against Salmonella Typhimurium (ST). FIG. 7 shows that, in Example 5, mice that were orally infected with ST after administration of the YS-1 / IL18 strain were inoculated with the YS-1 strain (parent strain) in the spleen, mesenteric lymph nodes, and Peyer's patch. It is a graph which shows having suppressed significantly compared with the control mouse | mouth which carried out. FIG. 8 shows that in Example 6, swine orally administered with the KO / IL18 strain, compared with control pigs orally inoculated with the parent strain (Koganei strain 65-0.15), swine erysipelas in the serum and bronchoalveolar lavage fluid Antigen SpaA. It is a graph which shows that the antibody titer with respect to 1 is rising.

  Embodiments of the present invention will be described below.

  The porcine vulgaris of the present invention is a porcine vulgaris that is a transformant of a attenuated strain of porcine vulgaris, has a recombinant DNA combined with a gene encoding a cytokine, and can express the cytokine. .

  The applicant has previously cloned the gene for Surface Protective Antigen (SpaA.1), which encodes the surface antigen of swine erysipelas, together with the Institute for Microbial Chemistry, and removed a part of this gene SpaA.1. A chimeric gene was constructed by inserting part of the P97 adhesin gene of Mycoplasma hyopneumoniae, a foreign gene, and the chimeric gene was introduced into swine gonococci to develop swine erysipelas that express proteins derived from this chimeric gene. (Japanese Patent Laid-Open No. 2002-119285).

  In the present invention, by applying the above technique, a gene encoding cytokine is used as a foreign gene, a chimeric gene in which a gene encoding cytokine is inserted into a part of SpaA.1 is prepared, By introducing it into an attenuated strain, a transformant of swine erysipelas expressing cytokines is produced.

  Examples of attenuated strains of swine erysipelas used as a host in the present invention include attenuated strains of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum, and are often used in Japan, for example. Derived from a transposon mutant strain of the swine genococcal strain Fujisawa-SmR described in S. cerevisiae Koganei strain 65-0.15 and patent publication No. 299980 previously filed by the present applicant, Examples include erythrocyte capsular deletion mutant strain (FERM P-16466) showing sensitivity. All of the attenuated strains of swine erysipelas used as the host are attenuated to pigs, and the live bacteria do not cause disease even when infected with animals other than pigs. Among them, from the viewpoint of easy handling, commercially available swine erysipelas Koganei strain 65-0.15 and capsular deficient swine erysipelas attenuated YS-1 strain (FERM P-16466) are preferred.

  In the present invention, the cytokine is at least one selected from the group consisting of interleukin (IL), interferon (IFN), hematopoietic factor, cell growth factor and cytotoxic factor. IL is IL-1, IL-2, IL-4, IL-18, IL-8, etc., and IFN is IFN-α, IFN-β, IFN-γ, IFN-ε, INF-κ, etc. Hematopoietic factors include colony-stimulating factor (CSF), granulocyte colony stimulating factor (G- CSF), and erythropoietin (Erythropoietin (EPO)). Examples of cytotoxic factors such as EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and transforming growth factor (TGF) include tumor necrosis factor (TNF-α). ), Lymphotoxin (TNF-β) and the like.

  Examples of the animal that is the origin of the cytokine include various animals such as humans, dogs, cats, cows, pigs, sheep, birds, dolphins, etc., but there is no particular limitation as long as the animal is intended to enhance immunity. .

  Cloning of the gene encoding the cytokine is performed using primers selected based on DNA information of a desired cytokine obtained from a database such as DDBJ / GenBank / EMBL after extraction and purification of mRNA from various raw materials according to a conventional method. What is necessary is just to carry out by PCR reaction using cDNA synthesized by a photoenzyme as a template. The gene product containing the gene encoding the cytokine amplified by the PCR is prepared so as to have a gene sequence having EcoRI cleavage sites upstream and downstream of the gene encoding the cytokine. In order to use the later-described chimeric gene for production, the gene product may be fragmented by enzymatic treatment with EcoRI in advance.

  In the present invention, as a recombinant DNA to which the gene encoding the cytokine is bound, according to the method described in JP-A No. 2002-119285, the gene encoding the cytokine is converted to SpaA. A chimeric gene designed so as to be sandwiched between the center of one gene is prepared.

  SpaA. Used for the production of the chimeric gene. One gene includes spaA.I described in JP-A No. 2002-119285. Phagemid pERc6.1 containing the full length of one gene is used.

  The phagemid pERc6.1 was prepared as follows. Specifically, as already reported by Inventor, who is the present inventor (Infection and Immunity 1999, p1646-1651), the chromosomal gene of swine erysipelas fujisawa strain was determined by the method of Galan and Timoney et al. (Infection and Immunity 1990 , 58, p3116-3121), partially digesting this chromosomal gene with the restriction enzyme Sau3AI, fractionating 3-5 kilobase pairs of gene fragments by cyclosgradient centrifugation, and obtaining the obtained gene fragments Ligated to the BamHI site of Zap Express Vector (Stratagene: La Jolla, Calf.), In vitro packaging using Gigapack III Gold packaging extract (Stratagene), and infecting the resulting recombinant phage with E. coli To make phage plaques on agar plates. This phage plaque is transferred to a nitrocellulose membrane previously impregnated with IPTG (isopropylthiogalactoside), reacted with serum obtained from pigs experimentally infected with swine erysipelas highly virulent strain, and reacted by an enzyme antibody method. Was detected. The swine erysipelas gene cloned in the selected recombinant phage was expressed in E. coli, the expressed protein was purified, and mice were immunized. After immunization, he attacked with highly virulent swine erysipelas Fujisawa strain. Recombinant phage clones expressing proteins that showed protection against this attack were finally selected. Furthermore, the phagemid pERc6 was produced from the selected clone using ExAssist helper phage E. coli XLOLR system (Stratagene). The inserted gene sequence of phagemid pERc6 was determined by the cycle sequence method, and this sequence was compared with the sequence registered in Genebank. As a result, the surface protective protein (Surface protective antigen: SpaA) reported by Makino et al. ) And high homology. However, since the sequence of Makino et al. Differs from the C-terminal sequence in particular, this gene was designated spaA. It was set to 1. Phagemid pERc6 was subcloned and spaA. A phagemid (plasmid) pERc6.1 containing the full length of one gene was prepared.

In the present invention, the chimeric gene can be prepared as follows.
That is, the SpaA. Since there are two restriction enzyme EcoRI sites in one gene, plasmid pERc6.1 is treated with restriction enzyme EcoRI. Next, a gene product containing a gene encoding a cytokine (a gene fragment having a restriction enzyme EcoRI cleavage site at both ends) and the prepared plasmid pERc6.1 are ligated by a conventional method, transformed into Escherichia coli, and a chimeric gene. E. coli carrying the plasmid is obtained. This plasmid is SpaA. Since a promoter sequence is included on the N-terminal side of one gene, the protein can be expressed in Escherichia coli without the addition of a commonly added drug such as IPTG. By analyzing the protein expressed with the growth of E. coli by Western blotting, the molecular weight of the expressed protein, and SpaA. The reaction of both the monoclonal antibody against 1 and the monoclonal antibody against cytokine can be confirmed. This SpaA. A monoclonal antibody against 1 is an antibody having a protective ability against passive infection. From this result, it is possible to confirm the expression of a chimeric protein in which the Porphylococcus spa A protein and cytokine are fused, and in addition, a gene cassette related to the expression of this protein can be prepared.

Next, according to the method described in JP-A No. 2002-119285, the chimera gene is transformed into an attenuated strain of swine erysipelas by the following procedure.
That is, a PCR primer having a restriction enzyme ClaI site for amplifying the entire insert of a plasmid, which is a chimeric gene, is designed on the 5 ′ side, and PCR is performed using the primer and T7 primer. The amplified product is treated with the restriction enzyme ClaI and ligated to the Streptococcus suis previously treated with the restriction enzyme ClaI and the shuttle vector pGA14 of Escherichia coli. This plasmid is introduced into a attenuated strain of swine erysipelas by electroporation.
Among the transformants, spaA. As a result of a double crossover replacing the upstream / downstream region of one gene with the introduced gene, that is, the homologous region on the pER6.1 / IL18 plasmid, a strain that does not show drug resistance is selected. Confirm that you have the gene of interest.
The method for producing a competent cell used for the transformation method, the conditions of the electroporation method used for the transformation, and the method for selecting the transformant were the methods already reported by Shimoji et al. Infection and Immunity, 70: 226-232, 2002).

The porcine erysipelas of the present invention obtained as described above can be easily propagated at low cost by using conventional culture means. Further, when the swine venom of the present invention is administered to a non-human animal, it can settle in each part of the non-human animal body, and a cytokine can be administered to each part.
Furthermore, as long as the swine venom of the present invention is alive, it is possible to continue to supply cytokines to the established non-human animal body. Is done.
Therefore, the present invention is a simple and safe cytokine delivery system to non-human animals in that it is not necessary to administer purified cytokine every time it is needed.

  Examples of routes for administering the porcine erysipelas of the present invention to non-human animals include oral administration, nasal administration, enema administration, etc. Oral administration is preferred from the viewpoint of easy operation. For example, the swine venom itself or a liquid in which the cells are suspended in water or the like can be used.

As described above, in the body of a non-human animal that has been administered swine erysipelas, an immunopotentiating effect is exerted by the safe supply of cytokines.
Therefore, administration of swine erysipelas to non-human animals makes it difficult to suffer from diseases such as infectious diseases, reduces symptoms, and further cures diseases earlier by using other vaccine preparations in combination. be able to. In addition, since the swine venom of the present invention is considered not to have a great influence even when mixed with a vaccine for non-human animals, for example, the swine venom of the present invention is contained in a vaccine preparation for non-human animals. be able to.

  Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

(Example 1: Production of capsular-deficient swine erysipelas attenuated YS-1 strain (YS-1 / IL18 strain) into which poIL-18 gene was introduced)
According to the method described in JP-A No. 2002-119285, a capsular membrane in which a porcine interleukin-18 (poIL-18) gene was introduced as a foreign gene to be introduced in place of the P97 adhesin gene, which is an adhesion protein of Mycoplasma hyopneumoniae A deficient porcine erysipelas attenuated YS-1 strain (YS-1 / IL18 strain) was prepared.

  In addition, as the poIL-18 gene, plasmid DNA (pVL1392-IL18) containing a gene encoding porcine IL-18 described in Japanese Patent No. 3541216 filed by Shimo et al., The present inventor, was used. A DNA fragment excised from the PCR product of plasmid DNA with EcoRI (a gene encoding porcine IL-18 after caspase cleavage recognition amino acid sequence LESD) was used.

All cloning and analysis technique, standard protocols (Sambrook J., EF Fritsch, and T.Maniatis 1989. Molecular cloning:.. A Laboratory manual, 2 nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) Went according to.

  In addition, the method for producing a competent cell used for the transformation method, the conditions of the electroporation method used for the transformation, and the method for selecting the transformant were the methods already reported by Shimoji et al. Infection and Immunity, 70: 226-232, 2002).

Among the transformants, spaA. As a result of double crossover replacing the upstream / downstream region of one gene with the introduced gene, that is, the homologous region on the pER6.1 / IL18 plasmid, a strain that showed no drug resistance was selected. Confirmed that it possesses the target gene. This strain was designated as YS-1 / IL18 strain.
In addition, in FIG. 1, the procedure of preparation of the porcine erysipelas YS-1 / IL18 strain | stump | stock is shown.

The YS-1 / IL18 strain was cultured overnight at 37 ° C. in a culture medium (composition: brain heart infusion supplemented with 0.1% Tween 80), centrifuged (× 9,000 g), and cultured. I got Qing. Next, the resulting bacterial cells were mixed with a buffer (composition: phosphate buffered saline containing 0.1% Tween 20), crushed by sonication, and centrifuged to centrifuge the microbial cells. Got.
The absorbance at OD 450 nm of the culture supernatant and the disrupted cell supernatant was measured according to the method described in JP-A-2001-103967, and the concentration of poIL-18 contained in these supernatants was measured. .
In addition, as a control, the culture supernatant of the YS-1 strain (parent strain) that was not transformed and the centrifuge supernatant of the microbial cells were also examined. These results are shown in FIG.
From the results shown in FIG. 2, in the YS-1 strain (parent strain), poIL-18 is hardly seen in either the culture supernatant or the disrupted cell supernatant, whereas in the YS-1 / IL18 strain, It can be seen that poIL-18 is remarkably expressed in both the culture supernatant and the disrupted cell supernatant.

(Example 2: Production of 65-0.15 strain of porcine erysipelas introduced with poIL-18 gene, Koganei strain (KO / IL18 strain))
Swine erysipelas introduced with the poIL-18 gene in the same manner as in Example 1 except that the capsular-deficient swine venom attenuated YS-1 strain was used as the host cell. Bacteria Koganei 65-0.15 strain (KO / IL18 strain) was prepared. The same DNA fragment as in Example 1 was used as the poIL-18 gene.

  Among the transformants, spaA. As a result of double crossover replacing the upstream / downstream region of one gene with the introduced gene, that is, the homologous region on the pER6.1 / IL18 plasmid, a strain that showed no drug resistance was selected. Confirmed that it possesses the target gene. This strain was designated as KO / IL18 strain.

In addition, the KO / IL18 strain was cultured overnight at 37 ° C. in a culture medium (composition: brain heart infusion supplemented with 0.1% Tween 80), centrifuged (× 9,000 g), and the culture supernatant. Got. Next, the resulting bacterial cells were mixed with a buffer (composition: phosphate buffered saline containing 0.1% Tween 20), crushed by sonication, and centrifuged to centrifuge the microbial cells. Got.
The absorbance at OD 450 nm of the culture supernatant and the crush supernatant of disrupted cells was measured, and the concentration of poIL-18 contained in these supernatants was measured.
In addition, as a control, the culture supernatant of the Koganei 65-0.15 strain (parent strain) that was not transformed and the centrifuge supernatant of the microbial cells were also examined. These results are shown in FIG.
From the results shown in FIG. 3, in the Koganei 65-0.15 strain (parent strain), almost no poIL-18 was observed in either the culture supernatant or the cell disruption centrifuge supernatant, whereas the KO / IL18 strain Then, it can be seen that poIL-18 is remarkably expressed in both the culture supernatant and the microbial cell centrifuge supernatant.

(Example 3)
Spleen cells were removed from 2 mice (10 weeks old), adjusted to 2 × 10 ⁶ / ml with RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, and then applied to 100 μl of this cell suspension. 100 μl of a 2-fold dilution of the culture supernatant of the YS-1 / IL18 strain obtained in Example 1 was added and cultured overnight at 37 ° C., and production of IFN-γ was performed using a mouse IFN-γ ELISA kit (Invitrogen). The amount was examined. As a control, the production amount of IFN-γ was similarly examined using the culture supernatant of the YS-1 strain (parent strain). The results are shown in FIG.
From the results shown in FIG. 4, IFN-γ production is below the detection limit in the culture supernatant of the YS-1 strain (parent strain), whereas in the culture supernatant of the YS-1 / IL18 strain, IFN-γ. Production was confirmed.

Moreover, about the induction | guidance | derivation ability of YS-1 / IL18 stock | strain and YS-1 stock | strain (parent strain) of YS-1 / IL18 strain or YS-1 stock | strain (parent strain) 1.3 * 10 < ⁸ > CFU bacteria are included. Brain heart infusion (BHI) medium (Becton, Dickinson and Company (BD)) was intraperitoneally administered to 7 mice (9 weeks old), and the spleen cells of the mice were collected on the 5th day. It adjusted to x10 < ⁶ > / ml piece. To 100 μl of the cell suspension, 100 μl of concanavalin A solution (5 μg / ml) was added and cultured overnight at 37 ° C., and then the IFN-γ in the culture supernatant was assayed using a mouse IFN-γ ELISA kit (Invitrogen). The amount was examined. The result is shown in FIG.
From the results shown in FIG. 5, the YS-1 / IL18 strain is significantly more significant in the mouse than the YS-1 strain (parental strain), because the YS-1 / IL18 strain is established in the body of the mouse and IL18 is supplied. It can be seen that the production of IFN-γ is promoted.

  From the above results, it can be seen that the porcine erysipelas of the present invention can enhance immunity of mice that are non-human animals.

Example 4
32 mice (7 weeks old) were intraperitoneally administered with a BHI medium containing 1.3 × 10 ⁷ CFU / animal strain of YS-1 / IL18 strain obtained in Example 1. As a control, the same number of mice were inoculated intraperitoneally with 1.0 × 10 ⁷ CFU / animal of YS-1 strain (parent strain). On day 5, 1 week, 2 weeks, and 3 weeks after administration of the fungus, 8 mice of each fungus administration group were euthanized, and using macrophages collected from the peritoneal cavity of the mice, Salmonella Typhimurium was used. The phagocytic ability for (ST) was examined. Phagocytosis was measured by dropping macrophages one hour after phagocytosis on the medium and counting the colonies of the bacteria that appeared. The results are shown in FIG.
From the results shown in FIG. 6, the peritoneal macrophages of the mice administered with the YS-1 / IL18 strain were compared with the macrophages of the mice inoculated with the YS-1 strain (parent strain). , It was confirmed that the phagocytic ability for ST was enhanced.
From this, as can be seen from the fact that the phagocytic ability of macrophages was enhanced in mice administered with the YS-1 / IL18 strain, the YS-1 / IL18 strain was established in the body of the mouse and IL18 was supplied. It can be seen that immunity is non-specifically activated.
During the above experiment, mice were raised in a room where temperature and humidity were controlled according to a conventional method, and food and water were given freely.

(Example 5)
5 days after intraperitoneal administration of BHI medium containing 1.3 × 10 ⁷ CFU / animal of the YS-1 / IL18 strain obtained in Example 1 to 5 mice (10 weeks old) Was orally inoculated with Luria-Bertani medium (BD) containing 1.0 × 10 ⁹ CFU / head of bacteria, and the mice were euthanized on the 19th day, and the spleen, mesenteric lymph nodes and Peyer's patch were examined in a conventional manner. The number of STs present in each organ was measured by dropping tissue homogenate onto the medium and counting the colonies of the bacteria that appeared.
Further, as a control, the number of STs in each organ was similarly measured using a YS-1 strain (parent strain) 1.2 × 10 ⁷ CFU / animal. The results are shown in FIG.
From the results shown in FIG. 7, the number of Salmonella present in the three types of organs was significantly lower when the YS-1 / IL18 strain was inoculated than when the YS-1 strain (parent strain) was used. It was. In particular, since the number of Salmonella present in the mesenteric lymph nodes and Peyer's patch was remarkably low, in the mice administered with the YS-1 / IL18 strain, the YS-1 / IL18 strain was established in the body and IL18 was supplied. As a result, it can be seen that, as a result of activation of immunity, the ability to eliminate Salmonella is enhanced.
Therefore, it can be seen that the porcine erysipelas of the present invention is effective for the prevention and / or treatment of diseases of mice that are non-human animals.
During the above experiment, mice were raised in the same manner as in Example 4.

(Example 6)
The artificial milk supplemented with 7 × 10 ¹⁰ CFU / head of the KO / IL18 strain obtained in Example 2 so as to be 7 × 10 ¹⁰ CFU / head was orally administered to two aseptic pigs (produced by DOG Lab). On day 13 after administration of the bacterium, SpaA. The antibody concentrations of IgA and IgG against one antigen were measured.
In addition, as a control, using two sterile pigs administered with the KO strain (parent strain), SpaA. Antibody concentration against one antigen was measured by ELISA. The results are shown in FIG.
From the results shown in FIG. 8, in the pigs orally administered with the KO / IL18 strain, the porcine erysipelas antigen SpaA.I in the serum and bronchoalveolar lavage fluid (BALF) was compared with the control pig inoculated orally with the KO strain (parent strain). The antibody titer against 1 (IgA, IgG) was increased.
From this, in pigs that were orally administered with the KO / IL18 strain, the KO / IL18 strain was established in the body and IL18 was supplied, and as a result, the immunity of the pig was activated, resulting in increased antibody production. It can be seen that there is an adjuvant effect.
Therefore, the porcine erysipelas of the present invention can be used as a vaccine adjuvant in non-human animals. In this case, a vaccine preparation for non-human animals can be prepared by using the porcine erysipelas of the present invention instead of an adjuvant in the vaccine preparation.
In addition, even when the porcine erysipelas of the present invention is used alone, it can be seen that it is effective for the prevention and / or treatment of diseases of pigs that are non-human animals.
During the above experiment, pigs were raised in a room where temperature and humidity were controlled according to a conventional method, and water was given freely, but food was given once a day.

  In the above Examples 1 and 2, porcine IL-18 was used as a cytokine, but even when other porcine cytokines or cytokines derived from other animals were used, according to the procedure described in Examples 1 and 2, It is possible to produce transformed strains of the A. vulgaris attenuated strain YS-1 and KO strain that produce the desired cytokine.

Claims (7)

  A swine gonococcus, which is a transformant of a porcine erysipelas attenuated strain, has a recombinant DNA combined with a gene encoding a cytokine, and expresses the cytokine.   The swine erysipelas according to claim 1, wherein the cytokine is at least one selected from the group consisting of interleukins, interferons, hematopoietic factors, cell growth factors and cytotoxic factors.   The swine venom of claim 1 or 2, wherein the swine venom attenuated strain serving as the host is a swine venom koganei strain 65-0.15 or a capsular-deficient swine venom attenuated strain YS-1.   A method for preventing and / or treating a disease in a non-human animal, comprising a step of administering the swine erysipelas according to claim 1 to a non-human animal.   A delivery system for cytokines into a non-human animal body, wherein the swine venom of any one of claims 1 to 3 is administered to a non-human animal.   A method of using the porcine erysipelas according to any one of claims 1 to 3 as a vaccine adjuvant in a non-human animal.   The vaccine formulation for non-human animals containing the porcine erysipelas in any one of Claims 1-3.

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