WO2009113665A1

WO2009113665A1 - Method of producing drug for swine atrophic rhinitis

Info

Publication number: WO2009113665A1
Application number: PCT/JP2009/054889
Authority: WO
Inventors: 透河合; 顕治横川; 容子本田
Original assignee: 財団法人化学及血清療法研究所
Priority date: 2008-03-13
Filing date: 2009-03-13
Publication date: 2009-09-17
Also published as: JP2011116658A

Abstract

Provided is a method of producing a drug for swine atrophic rhinitis. A method of producing a drug for swine atrophic rhinitis comprising: (1) a step of culturing Bordetella bronchiseptica dermonecrotic toxin-producing Escherichia coli which is obtained by a transgenic technique; (2) a step of collecting and purifying the dermonecrotic toxin which is expressed in a soluble state from a suspension of disrupted cells of the strain as described in (1); (3) a step of detoxifying the dermonecrotic toxin as described in (2) with formalin; and (4) a step of producing a preparation of the detoxified dermonecrotic toxin as described in (3).

Description

Method for producing a drug for swine atrophic rhinitis

The present invention relates to a method for producing a drug for swine atrophic rhinitis. Specifically, a step of culturing a skin necrotic toxin-producing host obtained by a genetic recombination technique, a step of recovering and purifying soluble skin necrotic toxin from the culture, and a step of detoxifying the skin necrotic toxin And a method for producing a drug for swine atrophic rhinitis, comprising the step of formulating the detoxified skin necrosis toxin.

Porcine atrophic rhinitis (hereinafter sometimes abbreviated as AR) is a respiratory disease characterized by turbinate dysplasia or atrophy. This is a serious disease for the pig farming industry because it causes serious economic loss due to growth delay, induction of secondary infection, and the like (see, for example, Non-Patent Document 1). As its causative bacteria, Bordetella bronchiseptica (hereinafter sometimes abbreviated as Bb) and toxin-producing Pasteurella multocida (hereinafter also abbreviated as Pm) are known. Bb possesses hemagglutinin and fimbriae, which are fixing factors, and thus has a strong fixability to the nasal mucosa, but Pm that does not have it is difficult to settle alone. Therefore, the presence of a factor that damages the nasal mucosa in advance is necessary for the formation of toxin-producing Pm infection. The most representative factor is skin necrosis toxin (hereinafter abbreviated as Bb-DNT) produced by Bb. It is believed that toxin-producing Pm settles in the damaged nasal mucosa and that the symptoms of AR are exacerbated by the toxin produced (hereinafter abbreviated as PmT).

Bb-DNT has biological activities such as bleeding necrosis, nasal turbinate atrophy, growth delay, vascular smooth muscle contraction, local blood circulation disorder, spleen atrophy and antibody production inhibition. Therefore, the necessity of a vaccine in which Bb-DNT is toxoidated has been pointed out for a long time (see, for example, Non-Patent Document 2). So far, methods using ion exchange chromatography (for example, see Non-Patent Document 3), methods using dialysis, sucrose density gradient ultracentrifugation and gel filtration chromatography (for example, see Non-Patent Document 4), nucleic acid removing agents and Attempts were made to produce a toxoid vaccine for Bb-DNT purified by a method such as a combination of ion exchange chromatography (for example, see Non-Patent Document 5). It hasn't arrived. The only toxoid vaccine of Bb-DNT purified by a method using a chromatographic carrier gel into which a sulfate group has been introduced (see, for example, Patent Document 1) has been successfully commercialized. Currently, in order to prevent AR, Bb killed vaccine is used as a simple inactivated vaccine, component vaccine based on fibrous hemagglutinin produced by Bb and PmT toxoid, and Pm death is used as a combined vaccine. Bacteria and Bb killed mixed vaccine, PmT toxoid and Bb killed mixed vaccine, Bb-DNT toxoid and PmT toxoid mixed vaccine, etc. have been put into practical use. In any case, a toxoid chemically inactivated with formalin or the like is used.

The expression of useful proteins using genetic recombination techniques is a widely used technique today, and among them, expression systems using E. coli as the host are the most commonly used expression systems. For expression of useful proteins, it is common to construct an expression vector in which a target gene is bound downstream of a promoter sequence. For example, lac, trp, tac, trc, ara, T7, etc. are known as promoter sequences of E. coli. However, it is known that when a useful protein is expressed at a high level using Escherichia coli, an insoluble inclusion body is often formed (for example, see Non-Patent Document 6). When obtaining an active protein from an inclusion body, generally, a method is employed in which the inclusion body is once solubilized using a protein denaturant such as urea or guanidine hydrochloride and then refolded.

Various methods have been tried for such solubilization and refolding, but the method varies greatly depending on the protein to be handled, and often reduces the recovery amount of the target active protein. Attempts have also been made to express Bb-DNT using E. coli. Pullinger et al. Determined the base sequence of Bb-DNT and reported expression in Escherichia coli using the trc promoter (see, for example, Non-Patent Document 7). Horiguchi et al. Have reported expression using the T7 promoter (see, for example, Non-Patent Document 8). However, in these reports, only the full length or a part of Bb-DNT is purified by recombinant E. coli and its activity is evaluated. The industrial use of these recombinants, for example, expression of the recombinants There is no indication of the amount, the toxin activity of the insoluble fraction, or the usefulness as a vaccine using it.
Japanese Patent No. 3884066 Amagasaki et al., Hog Pathology 4th Edition, pp.286-294, 1999 Roop II et al., Infect. Immun., 55: pp. 217-222, 1987 Kume et al., Infect. Immun., 52: pp.370-377, 1986 Endoh et al., Microbiol. Immun., 30: pp. 659-673, 1986 Horiguchi et al., FEMS Microbiol. Lett., 66: pp.39-44, 1990 Singh et al., J. Biosci. Bioeng., 99: pp.303-310, 2005 Pullinger et al., Infect. Immun., 64: pp.4163-4171, 1996 Horiguchi et al., Proc. Natl. Acad. Sci. USA., 94: pp. 11623-11626, 1997

As mentioned above, although dead germ vaccines and component vaccines for preventing AR have been put into practical use and have greatly contributed to the pig farming industry, there is room for improvement and improvement in cost, safety measures, and vaccine quality improvement. Therefore, the problem to be solved by the present invention is a drug for swine atrophic rhinitis which contains recombinant skin necrosis toxoid as an active ingredient, which is effective in preventing the onset of AR and satisfies safety, low cost and stable supply. It is in providing the manufacturing method of. Here, the “drug” in the present invention is defined as a pharmaceutical composition applied for treatment and prevention.

As a result of intensive studies to achieve the above object, the present inventors have referred to recombinant Escherichia coli (hereinafter referred to as Bb-DNT-producing Escherichia coli) that produces Bordeterra spp. Skin necrosis toxin (Bb-DNT). And Bb-DNT was recovered from the Bb-DNT-producing Escherichia coli, and it was observed that Bb-DNT was recovered from both the soluble and insoluble fractions. After immunizing mice with the obtained soluble and insoluble fractions of skin necrosis toxin, an attack test was carried out with an active form of Bordetella sp. Only mice immunized with necrotic toxin have been found to be free from lethality, and the present invention has been completed.

That is, the present invention is as follows.
[1] A method for producing a drug for swine atrophic rhinitis, comprising a step of recovering recombinant skin necrosis toxin solublely expressed from a skin necrosis toxin producing host.
[2] A method for producing a drug for swine atrophic rhinitis comprising the following steps (1) to (4):
(1) a step of culturing a skin necrotic toxin-producing host obtained by a genetic recombination technique,
(2) recovering and purifying soluble necrotic toxin from the culture of (1),
(3) a step of detoxifying the skin necrosis toxin of (2),
(4) The process of formulating the detoxified skin necrosis toxin of (3) [3] The production method of [1] or [2], wherein the skin necrosis toxin is derived from Bordetella bacteria.
[4] The production method according to [3], wherein the Bordetella is selected from the group consisting of Bordetella bronchiseptica, Bordetella pertussis, and Parapertussis.
[5] The production method according to any one of [1] to [4], wherein the host is Escherichia coli.

According to the present invention, a method for producing a drug for swine atrophic rhinitis that is effective in preventing the onset of AR is provided. The method for producing a drug for swine atrophic rhinitis of the present invention includes a step of recovering soluble necrotic toxin from crushed Bb-DNT-producing Escherichia coli. Only the skin necrosis toxin recovered by this step is recovered as an antigenic protein having immunogenicity capable of preventing the onset of AR and can be used for subsequent formulation.

The feature of the present invention resides in a method for producing a drug for swine atrophic rhinitis including a step of recovering soluble skin necrosis toxin expressed in a soluble manner from a skin necrosis toxin production host.

The object of the production method of the present invention is to form an insoluble inclusion body when expressed by genetic recombination technology, and to prevent the onset of AR even if it is solubilized with a protein denaturant such as guanidine hydrochloride or urea. It is a skin necrotic toxin that cannot obtain effective immunogenicity. Examples of such skin necrosis toxin include Bordeterra bacteria-derived skin necrosis toxin, preferably Bordetella bronchiseptica skin necrosis toxin (Bb-DNT), and pertussis skin necrosis that can be used as an alternative to AR vaccine. Toxin (Bp-DNT) (see JP-A-10-251298) and B. pertussis skin necrosis toxin with 99% homology to the base sequence of Bb-DNT (hereinafter sometimes referred to as Bpp-DNT) (See Non-Patent Document 6, Kimberly E Walker and Alison Ann Weiss Infection and Immunity 62, 3817-3838, 1994). Below, the manufacturing method of the chemical | medical agent for swine atrophic rhinitis using Bb-DNT is explained in full detail.

(1) Cloning of Bordetella spp. Skin necrosis toxin gene In this example, Bb-DNT cloned from Bordetella bronchiceptica S611 strain isolated and maintained from field materials in 1988 at the Institute of Chemical and Serum Therapy The encoding gene was used. For the growth of Bordetella bronchiseptica, for example, McConkie medium (Nippon Becton Dickinson), Borde Jung medium (Nippon Becton Dickinson), Horiguchi et al. (Horiguchi et al., Microb. Pathog., 6 : 361-368, 1989). What is necessary is just to select suitably according to the objectives, such as isolation | separation of bacteria, preparation of a master seed, and mass culture. In the examples of the present application, the medium of Horiguchi et al. Was used for the growth of small and medium-sized cells. Before use, the pH is adjusted to 6.8-7.6 according to the attached protocol, followed by autoclaving at 115 ° C for 25 minutes. The culture conditions are usually set in the range of a temperature of 36 to 38 ° C. and a period of 1 to 5 days, but may be appropriately adjusted according to the purpose of use, culture form, amount of planted bacteria, medium scale and the like.

The bacterial cells in the culture solution are collected in the sediment by low-speed centrifugation (5000 rpm, 5 to 10 minutes). The gene encoding Bb-DNT of Bordetella bronchiceptica is derived from the general DNA recombination technology described by Sambrook et al. (Molecular Cloning, A Laboratory Manual Second Edition. Cold Spring Harbor Laboratory Press, NY, 1989). In practice, various commercially available kits are used. For example, to acquire genomic DNA, ISOPLANT (Wako Pure Chemical), Instagene (Nippon Bio-Rad Co., Ltd.), EZNA Bacterial DNA Kit (Funakoshi Co., Ltd.), MagPrep Bacterial Genomic DNA Kit (Takara Bio Inc.), DNA Kits such as isolation kits (Roche Diagnostics Co., Ltd.) and QIAamp DNA Mini Kit (Qiagen Co., Ltd.) are used, and Takara Ex using the restriction enzyme digestion method and PCR method for gene acquisition Taq (Takara Bio Inc.), iTaq DNA Polymerase (Nippon Bio-Rad Co., Ltd.), KOD DNA Polymerase (Toyobo Co., Ltd.), Taq DNA Polymerase (Qiagen Co., Ltd.), etc. are used. Moreover, when cloning the obtained gene, kits such as TOPO-TA cloning kit (Invitrogen Corporation), pT7 BlueT-Vector (Takara Bio Inc.), QIAGEN PCR Cloning Kit (Qiagen) are used.

More specifically, chromosomal DNA is extracted from bacterial cells collected by low-speed centrifugation using ISOPLANT (Wako Pure Chemical Industries), and LA Taq (Takara Bio Inc.) is used as a template (template). The gene region encoding Bb-DNT is amplified by PCR according to the attached protocol. Primers used for PCR are designed based on the nucleotide sequence registered in GenBank Accession No. U59687. At this time, base sequences of appropriate restriction enzyme cleavage sites are added to the 5 'end of the upstream primer and the 5' end of the downstream primer. The PCR amplification product is cloned into a gene cloning vector, pCR-XL-TOPO (Invitrogen Corp.), and the nucleotide sequence is determined by a DNA sequencer (ABI Prism 310 Genetic Genetic Analyzer, Applied Biosystems). Thus, the plasmid pCRDNT into which the Bb-DNT gene has been inserted is obtained. The Bb-DNT gene of the present invention has the base sequence shown in SEQ ID NO: 1.

(2) Expression of Bb-DNT gene The Bb-DNT gene is expressed by excising the Bb-DNT gene from the above-described pCRDNT, incorporating it into an appropriate expression vector, and introducing the expression vector into a host. Bacteria, yeast, animal cells, plant cells, insect cells and the like are commonly used for the expression of foreign proteins. When expressing Bb-DNT toxoid, any host can be used, but bacteria such as Escherichia coli that are not toxic to the host are preferred. As expression vectors, various expression vectors having a promoter region such as trc promoter, T7 promoter, cspA promoter and the like have been developed and marketed for E. coli expression.

The Bb-DNT toxoid used as the antigen of the present invention does not retain immunogenicity capable of preventing the onset of AR when it is expressed insoluble. What is expressed as an insoluble inclusion body is once aggregated with a protein denaturant such as guanidine hydrochloride or urea, but easily aggregates when removed. Therefore, the Bb-DNT toxoid of the present invention is preferably expressed in a soluble manner. In the present invention, “to be expressed in a soluble manner” means that the target protein is expressed as a soluble protein in a state dissolved in Escherichia coli cells. As a method for soluble expression of Bb-DNT toxoid in E. coli, there are a method of culturing at a low temperature, a method of inducing expression with a low concentration of an inducing agent, a method of adding a secretory signal peptide and a method of coexpression with a chaperone protein. Any method may be used.

An appropriate Escherichia coli, such as BL21, HMS174, DH5α, HB101, JM109, etc., is selected as a host according to the expression vector. In this application example, a pCold vector (Takara Bio Inc.) having a cspA promoter capable of inducing protein expression at low temperature, a pTrc vector (GE Healthcare Bioscience Co., Ltd.) having a trc promoter, and a pET vector (Merck stock) having a T7 promoter Company). Transformation of E. coli can be performed using a commercially available competent cell according to the attached method. Thus, Escherichia coli ColdDNT, TrcDNT, and ETDNT producing Bb-DNT are obtained. As a medium (for example, LB, SOC, SOB, etc.) used for culturing Escherichia coli and a reagent (for example, ampicillin, etc.) used for selection of transformants, commercially available ones may be used. Further, the pH of the medium is used in a range suitable for the growth of E. coli (pH 6-8).

Screening of recombinant E. coli expressing Bb-DNT is performed as follows. If necessary, the cultured and proliferated cells in the presence of an appropriate expression inducer are collected by low-speed centrifugation, and a certain buffer (for example, 10 mM Tris (pH 8), 100 mM NaCl, 1 mM EDTA) is collected. After suspension, the cells are crushed with an ultrasonic crusher, French press, high-pressure homogenizer, etc., and separated and collected into a sediment or supernatant by high-speed centrifugation (15000 rpm, 15 minutes). A surfactant (eg, Triton® X100), lysozyme, etc. may be added to the buffer solution. A certain amount of Bb-DNT recovered in the supernatant and sediment is subjected to SDS-polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue, and then the expression of Bb-DNT protein is confirmed from the molecular size and stained image. Bb-DNT collected in the sediment is generally called an inclusion body. For confirmation (or detection) of Bb-DNT protein, methods based on antigen-antibody reactions such as ELISA, Western blot, and dot blot may be used in addition to the method based on the molecular size described above. Both are general methods for detecting foreign proteins expressed in E. coli, and may be appropriately selected according to the purpose.

When purifying these proteins from such Bb-DNT-producing Escherichia coli, purification methods generally used in protein chemistry, such as centrifugation, salting out, ultrafiltration, isoelectric precipitation, electrophoresis, etc. A method in which methods such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, hydrophobic chromatography, and hydroxyapatite chromatography are combined is used. Bb-DNT and Bb-DNT toxoids can be easily purified by cation exchange chromatography. The amount of the obtained protein is measured using a BCA Protein Assay Reagent Kit (Pierce Biotechnology, Inc), a protein assay kit (Nippon Bio-Rad Co., Ltd.) and the like.

(3) Evaluation of Bb-DNT toxoid as vaccine The thus obtained Bb-DNT is detoxified (toxoided) by various methods and evaluated as a vaccine. In general, formalin or glutaraldehyde is used for detoxification. The detoxification conditions are appropriately adjusted depending on the reagent used and the protein concentration. For example, the treatment is performed for 7 days at 37 to 40 ° C. using 0.8% formalin (Patent Document 1). Evaluation as a vaccine can be carried out by administering a lethal dose of Bb-DNT after immunization of a small animal. The immunization method (for example, subcutaneous, intramuscular, intraperitoneal, nasal, oral, sublingual administration site, immunization period, etc.) is generally in accordance with general methods used for examining immunogenicity of vaccines and the like. Just do it. As a positive control, a Bb cell disruption solution treated with formalin as described above is used. As a negative control, phosphate buffer solution, physiological saline, purified water or the like is used.

As an adjuvant, aluminum hydroxide, aluminum phosphate, mineral oil, non-mineral oil, etc. may be added to formalin-treated Bb-DNT. The Bb-DNT toxoid vaccine and control vaccine thus prepared were intraperitoneally administered intraperitoneally in groups of 5 to 10 mice, 2 weeks later, the second dose, and half the lethal dose after 2 weeks. Administer 10 times more Bb-DNT. Thereafter, the immunity of Bb-DNT toxoid is evaluated by observing the life and death of mice for 7 to 14 days. The Bb-DNT toxoid of the present invention can be an effective material as a vaccine for protecting against the onset of AR.

The Bb-DNT toxoid of the present invention contains additives generally used in addition to the above-mentioned adjuvant, such as stabilizers (arginine, polysorbate 80, macrogol 4000, etc.), excipients (mannitol, sorbitol, sucrose), etc. And then processed aseptic filtration, dispensing, lyophilization, etc., and formulated as an injection or transmucosally (nasally, orally, sublingually) to treat AR infection / onset Used as a vaccine to protect. The Bb-DNT toxoid of the present invention can also be used as a mixed vaccine for simultaneously protecting several types of swine infections by mixing with other swine infection vaccines. Such other swine infectious disease vaccines include swine Japanese encephalitis vaccine, swine infectious gastroenteritis vaccine, swine epidemic diarrhea vaccine, swine parvovirus infectious disease vaccine, swine getavirus infectious disease vaccine, swine oesky disease vaccine Swine erysipelas vaccine, swine reproductive / respiratory disorder syndrome vaccine, swine actinobacillus pleuropneumoniae infection vaccine, swine haemophilus paraswiss infection vaccine, swine E. coli diarrhea vaccine, swine mycoplasma hyopneumoniae infection vaccine, etc. Can be mentioned.
Hereinafter, the present invention will be specifically described with reference to examples.

<Cloning of Bordetella sp. Bb-DNT gene>
Bordetella bronchiseptica S611 strain (a strain isolated and maintained from field materials in 1988 at the Institute of Chemical and Serum Therapy) was used. This was inoculated into a medium of Horiguchi et al. (A composition table is shown in Table 1) and cultured at 37 ° C. for 1 day. The cells were collected by centrifugation, and chromosomal DNA was extracted using ISOPLANT (Wako Pure Chemical Industries). The medium of Horiguchi et al. Was adjusted to pH 6.8-7.6 and autoclaved at 115 ° C. for 25 minutes.

The Bb-DNT gene region was amplified by the PCR method using LA Taq (Takara Bio Inc.) using this chromosomal DNA as a template. The PCR primers used for amplification were designed from the nucleotide sequences registered in GenBank Accession No. U59687 (SEQ ID NOs: 2 and 3). A PciI site is added to the 5 'side of the amplified product, and a BamHI site is added to the 3' side. PCR was performed at 94 ° C for 60 seconds, followed by 20 times of 94 ° C, 30 seconds and 68 ° C for 5 minutes, and then allowed to react at 72 ° C for 10 minutes.

The PCR amplification product was mixed with pCR-XL-TOPO (Invitrogen Corporation), reacted according to the attached method, the reaction solution was added to TOP10F '(Invitrogen Corporation), and transformation was performed by a conventional method. Cultivation was carried out overnight at 37 ° C. in a kanamycin-added circle glow agar medium (Funakoshi Co., Ltd.), and a plasmid pCRDNT into which the Bb-DNT gene was inserted was obtained from the colonies that appeared. The base sequence of the Bb-DNT gene was determined by a DNA sequencer (ABI Prism 310 Genetic Analyzer, Applied Biosystems Japan Ltd.). The sequence is shown in SEQ ID NO: 1.

<Construction of expression vector>
The plasmid pCRDNT obtained in Example 1 was treated with restriction enzymes PciI and BamHI, and a Bb-DNT gene fragment of about 4.4 Kbp was purified. This was mixed with pTrc99A (Pharmacia Biotech Co., Ltd.) and pET-11d (Merck Co., Ltd.) previously treated with restriction enzymes NcoI and BamHI, and a ligation reaction was performed at 16 ° C. for 30 minutes (Ligation high, Toyobo Co., Ltd.) ). Each ligation reaction solution was added to DH5α competent cell (Takara Bio Inc.), and transformation was performed by a conventional method. Expression vectors pTrcDNT and pETDNT into which the Bb-DNT gene was inserted were obtained from colonies that had been inoculated into an ampicillin-added circle glow medium and appeared after overnight culture at 37 ° C.

In addition, the Bb-DNT gene region was amplified by the PCR method using LA Taq (Takara Bio Inc.) using pCRDNT obtained in Example 1 as a template. The PCR primers used for amplification were designed from the nucleotide sequences registered in GenBank Accession No. U59687 (SEQ ID NOs: 4 and 5). A restriction enzyme KpnI site is added to the 5 'side of the amplified product, and a restriction enzyme BamHI site is added to the 3' side. PCR was performed at 94 ° C for 60 seconds, followed by 20 times of 94 ° C, 30 seconds and 68 ° C for 5 minutes, and then allowed to react at 72 ° C for 10 minutes. The amplified fragment was treated with KpnI and BamHI, mixed with pColdIV (Takara Bio Inc.) previously digested with KpnI and BamHI, and ligated at 16 ° C. for 30 minutes. The ligation reaction solution was added to DH5α competent cells, and transformation was performed by a conventional method. An expression vector pColdDNT into which the Bb-DNT gene was inserted was obtained from colonies that had been inoculated into an ampicillin-added circle glow medium and appeared after overnight culture at 37 ° C.

<Expression of Bb-DNT>
E. coli BL21 strain (Merck Co., Ltd.) was transformed with each expression vector obtained in Example 2, and E. coli ColdDNT, TrcDNT and ETDNT producing Bb-DNT were cloned. A part of each clone was inoculated into 3 mL of ampicillin-supplemented YE medium (composition table is shown in Table 2) and cultured overnight with shaking. 50 μL of the bacterial solution obtained by shaking culture was inoculated into 50 mL of ampicillin-added YE medium and cultured at 37 ° C. with shaking. In the case of TrcDNT and ETDNT, when OD ₆₆₀ exceeds 1.0, IPTG (isopropyl-β-D-galactopyranoside) is added to a final concentration of 1 mM, and about 16 hours at 30 ° C. or 37 ° C. Continued shaking culture at. In the case of ColdDNT, when OD ₆₆₀ exceeded 1.0, it was allowed to stand at 15 ° C for 1 hour, and after IPTG was added, shaking culture was performed at 15 ° C for about 24 hours. After completion of the culture, the cells were collected by centrifugation, and the cells were suspended in a lysis buffer (50 mM Tris-Cl, 1 mM EDTA, 100 mM NaCl, pH 8.0). Lysozyme (Seikagaku Corporation, 10 mg / mL · DW) was added to the suspension, followed by lysis treatment at room temperature for 20 minutes. The lysis treatment solution was further sonicated and centrifuged, and the supernatant was used as a soluble fraction and the sediment was used as an insoluble fraction (inclusion body). Each fraction was subjected to SDS-PAGE according to a conventional method, and after CBB staining, the expression levels of Bb-DNT and Bb-DNT toxoid were calculated with a densitometer. The results are shown in Table 3.

《Bb-DNT toxoid vaccine evaluation》
Bb-DNT solublely expressed in ColdDNT obtained in Example 3 (sBb-ColdDNT) and Bb-DNT solublely expressed in TrcDNT (sBb-TrcDNT) were reported by Horiguchi et al. (Horiguchi et al., FEMS Microbiol. Lett., 66: 39-44, 1990). This was inactivated by adding formalin at a final concentration of 0.8% and treating at 37 ° C. for 7 days. Further, Bb-DNT (isBb-ETDNT) expressed insoluble in ETDNT obtained in Example 3 was dissolved in 8M urea solution. Vaccine solution containing Alhydrogel (Aluminum Hydroxide Gel, Brenntag Biosector A / S) to sBb-ColdDNT, sBb-TrcDNT and isBb-ETDNT solution, containing 1 ug of each antigen and 0.5 mg Alhydrogel per mL Was prepared. These three types of vaccines 0.5 mL were injected twice into the abdominal cavity of a 3-week-old female and SPF ddY mice, each 2 weeks apart. As a positive control, a commercially available AR vaccine (Swimgen ART2, Institute for Chemical and Serum Therapy) was used. This contains about 10 μg / mL of Bb-DNT purified and inactivated from the supernatant of the cell disruption solution by ultrasonic treatment of Bb S611 strain according to the method described in Japanese Patent No. 3886406 (Patent Document 1).

In order to adjust the dosage, alhydrogel and PBS were added, and a vaccine containing 1 ug of Bb-DNT and 0.5 mg of alhydrogel per 1 mL was prepared. As a negative control, a placebo vaccine containing 0.5 mg Alhydrogel per mL was prepared in PBS. Two weeks after the second administration, half the lethal dose of Bb-DNT (active form not treated with formalin inactivation) was injected intraperitoneally and observed for 7 days after injection. The results are shown in Table 4.

The production method of the present invention provides a swine atrophic rhinitis drug for preventing the onset of AR in pigs.

Claims

A method for producing a drug for swine atrophic rhinitis, comprising a step of recovering soluble skin necrotic toxin solublely expressed from a skin necrotic toxin producing host.
The manufacturing method of the chemical | medical agent for swine atrophic rhinitis including the process of following (1) to (4).
(1) a step of culturing a skin necrotic toxin-producing host obtained by a genetic recombination technique,
(2) recovering and purifying soluble necrotic toxin from the culture of (1),
(3) a step of detoxifying the skin necrosis toxin of (2),
(4) Step of formulating the detoxified skin necrosis toxin of (3)
The production method according to claim 1 or 2, wherein the skin necrosis toxin is derived from Bordetella bacteria.
The production method according to claim 3, wherein the Bordetella bacteria is selected from the group consisting of Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis.
The production method according to any one of claims 1 to 4, wherein the host is Escherichia coli.