Preparation and usage method of inactivated vaccine composition for progressive atrophic rhinitis

Abstract

The present invention belongs to the technical field of veterinary biological products, and specifically relates to a preparation and usage method of an inactivated vaccine composition for progressive atrophic rhinitis. The inactivated vaccine composition for progressive atrophic rhinitis comprises swine toxigenic Pasteurella multocida (ZXT+Pm), swine Bordetella bronchiseptica (AHBb), N-terminal and C-terminal proteins of toxigenic Pasteurella multocida toxin (PMT) (rPMT-N and rPMT-C) and a carbomer water adjuvant.

Description

Preparation and application method of a porcine atrophic rhinitis inactivated vaccine composition Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, in particular to a preparation and application method of a porcine atrophic rhinitis inactivated vaccine composition. Background Art

[0002] Progressive atrophic rhinitis (PAR) is a chronic respiratory disease characterized by widespread nasal dilation and atrophy of the mucosa, submucosa, and bone. It is highly contagious and is also known as infectious atrophic rhinitis or chronic atrophic rhinitis. It is recognized as a major infectious disease that seriously threatens the production and development of intensive pig farming. PAR is primarily caused by infection with toxin-producing Pasteurella multocida and Bordetella bronchiseptica, and often leads to secondary infections with other respiratory diseases such as mycoplasma, blue ear disease, and swine panting. The disease is widespread and occurs worldwide, with a relatively high incidence in developed pig farming regions and a high pathogen detection rate in pig herds. It is a major disease threatening large-scale pig farming and has been classified as a Category B disease by the World Organization for Animal Health. Therefore, the development of a more effective and safe inactivated vaccine for PAR is urgently needed.

[0003] Currently, porcine atrophic rhinitis is divided into progressive atrophic rhinitis and non-progressive atrophic rhinitis. Progressive atrophic rhinitis (PAR) is mainly caused by toxigenic strains (DNT+Pm) of Pasteurella multoeida (Pm) (mostly type D, a few type A); the other is non-progressive atrophie rhinitis (NPAR), which is mainly caused by phase I bacteria of Bordetella bronchiseptica (Bb). When the latter is infected alone, the nasal lesions are mild. When the two are mixed or secondary infections occur, the nasal lesions are very severe.

[0004] The pathogens of PAR are Bordetella bronchiseptica (Bb) and toxigenic Pasteurella multocida (Pm), both of the Bordetella genus. Bb is a Gram-negative, nonspore-forming, short rod with a peritrichous flagellum and motile. It possesses a capsule, is mostly bipolar, and is arranged in pairs or scattered. It can be divided into three phases: phase I, phase II, and phase III. Phase I bacteria are the most pathogenic. Typical phase I bacteria are pearly or semicircular, less than 1 mm in diameter, white, smooth, and dense, with indistinct β-hemolytic rings. Adding 5% to 10% sterile, defibrinated sheep whole blood to Bowden-Gang medium (BG) can maintain the colonies in phase I. Clinical studies have demonstrated that single Bordetella bronchiseptica infection is mild, and nasal bone defects can regenerate, representing a non-progressive atrophic rhinitis. Pasteurella multocida (Pm) is a Gram-negative bacterium with a spherical or short rod-shaped structure with blunt ends. Similar to Bb, Pm cannot form spores, but differs in that it is nonmotile. Based on lipopolysaccharide antigens, Pm strains can be divided into five capsular serogroups (A, B, D, E, and F) and 16 somatic serotypes (1 to 16). Serotypes A and D are among the pathogens that cause progressive porcine atrophic rhinitis. Serotypes A and D of P. multocida produce exotoxins. Research has shown that P. multocida possesses various virulence factors, including toxins, adhesins, and fimbriae. The most damaging is the dermonecrotic toxin (PMT), a protein approximately 146 kDa.

[0005] Symptoms of porcine atrophic rhinitis usually appear in pigs 3-4 weeks of age or after weaning, and the age of onset is negatively correlated with the severity of the disease. Sick pigs display varying degrees of catarrhal rhinitis, with frequent sneezing, difficulty breathing, and snoring. Piglets also exhibit decreased appetite and continuous serous or mucopurulent discharge from the nostrils, but the condition can return to normal when the disease is mild. In the late stages of infection, the nasal concha and trabeculae of sick pigs begin to atrophy and deform under the stimulation of inflammation. The nasal concha lesions extend from the ventral side to the dorsal side, the nostrils differ in size, and the nose tip tilts toward the affected side, resulting in the "crooked nose disease."

[0006] With the research on the pathogenic mechanism of porcine atrophic rhinitis, the best way to prevent the disease is vaccination. Currently, the commercial vaccines on the market are mainly inactivated bacterial vaccines and toxoid vaccines. It is found that the immune effect of toxoid vaccines is better than that of inactivated bacterial vaccines, but neither can achieve the desired effect. Therefore, this study used inactivated bacterial vaccines (ZXT + Pm and AHBb) plus recombinant Pasteurella multocida toxin proteins (rPMT-N and rPMT-C) can protect piglets from the attack of Bb and Pm on the one hand, and also prevent piglets from being attacked by T + The toxin protein produced by Pm has an osteolytic effect, thereby more comprehensively preventing and protecting piglets from porcine atrophic rhinitis.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a method for preparing and applying a porcine atrophic rhinitis inactivated vaccine composition to solve the problems raised in the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solutions: a preparation of a porcine atrophic rhinitis inactivated vaccine composition, the porcine atrophic rhinitis inactivated vaccine composition comprising porcine toxigenic Pasteurella multocida (ZXT + Pm), porcine bronchiseptica (AHBb), and toxigenic Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) with carbomer water adjuvant;

[0010] The specific steps of the preparation method of the porcine atrophic rhinitis inactivated vaccine composition are as follows:

[0011] Step 1: Store the porcine toxigenic Pasteurella multocida ZXT + The freeze-dried bacteria of strain Pm were inoculated into solid culture medium for resuscitation culture, and single clones were picked and cultured in liquid culture medium. After that, they were expanded in appropriate culture medium for 8-10 hours, and the bacterial liquid was harvested and inactivated.

[0012] Step 2: inoculating the preserved freeze-dried bacteria of Bordetella bronchiseptica AHBb strain into a solid culture medium for recovery culture, picking a single clone and culturing it in a liquid culture medium, then expanding the culture in a suitable culture medium for 10-12 hours, harvesting the bacterial solution, and inactivating it;

[0013] Step 3: The preserved engineered bacteria Rosetta-rPMT-N and Rosetta-rPMT-C freeze-dried bacteria were inoculated into solid culture medium for recovery culture, single clones were picked and cultured in liquid culture medium, and then expanded and induced for expression in a suitable culture medium. The bacteria were harvested by centrifugation and crushed under high pressure, and the target protein was harvested and purified. The N-terminal protein and C-terminal protein of toxin-producing Pasteurella multocida (rPMT-N and rPMT-C proteins) were harvested, sterilized, and the protein concentration was tested;

[0014] Step 4: Inactivate the porcine toxigenic Pasteurella multocida ZXT + The Pm strain, porcine bronchiseptica Pasteurella multocida AHBb strain, and toxigenic Pasteurella multocida N-terminal protein and C-terminal protein are mixed in a certain proportion, added with carbomer water adjuvant, and emulsified to prepare a polyvalent inactivated vaccine.

[0015] Preferably, the porcine toxigenic Pasteurella multocida (ZXT + Pm) and porcine bronchiseptica Bordetella swine (AHBb) were optimized and cultured until they entered the stationary phase, inactivated with formaldehyde, and treated with Triton X-114 to remove endotoxin.

[0016] Preferably, the toxigenic Pasteurella multocida PMT toxin N-terminal protein (rPMT-N) is 1461 bases from N1 to 1461, with a protein size of 88 kDa, and the C-terminal protein (rPMT-C) is 900 bases from C2959 to 3858, with a protein size of 67 kDa, and is cloned, expressed, and purified using Rosetta-rPMT-N and Rosetta-rPMT-C strains;

[0017] The process of cloning, expression and protein purification of the Rosetta-rPMT-N and Rosetta-rPMT-C strains is as follows:

[0018] (1) Amplifying the gene fragments shown in SEQ.NO.1 and SEQ.NO.2, ligating them into the linear vector pET32a to obtain pET32a-PMT-N and pET32a-PMT-C plasmids, and transforming the pET32a-PMT-N and pET32a-PMT-C plasmids into Escherichia coli Rosetta to obtain recombinant bacteria;

[0019] (2) Cultivating the recombinant bacteria obtained in step (1), then adding 1 mM IPTG to the culture medium, shaking at 30°C for 4 hours, and centrifuging to obtain bacterial cells;

[0020] (3) resuspending the microorganisms described in step (2) in PBS buffer, crushing them under high pressure, and collecting the supernatant by centrifugation to obtain a crude protein solution;

[0021] (4) Purifying the crude protein obtained in step (3) using a Ni column and eluting it with a certain concentration of imidazole for purification;

[0022] (5) After removing imidazole from the purified solution obtained in step (4), the concentration of the purified protein solution (rPMT-N or rPMT-C) was detected using a BCA kit.

[0023] Preferably, the porcine toxigenic Pasteurella multocida (ZXT + The contents of Pm) and AHBb were both 1×10 9 CFU / ml, and the content of the toxin-producing Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) is 10-80 μg / ml.

[0024] Preferably, the adjuvant is carbomer water adjuvant, which is mixed with the antigen component in an appropriate ratio.

[0025] Preferably, the base sequences of the N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the Pasteurella multocida-producing PMT toxin are SEQ.NO.1 and SEQ.NO.2, and the amino acid sequences are SEQ.NO.3 and SEQ.NO.4.

[0026] Preferably, the porcine toxigenic Pasteurella multocida (ZXT + Pm) and porcine bronchiseptica Bordetella suis (AHBb) content was 1×10 9 CFU / ml, the content of the toxin-producing Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) is 10-80 μg / ml, and is mixed with a certain proportion of carbomer water adjuvant.

[0027] A method for using the porcine atrophic rhinitis inactivated vaccine composition according to any of claims 1 to 7, and use of the porcine atrophic rhinitis inactivated vaccine composition in the preparation of a vaccine or medicine for preventing or treating progressive or non-progressive porcine atrophic rhinitis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Porcine atrophic rhinitis has many hosts and is widely distributed. The distribution of serotypes varies in different regions, and the cross-protection between different serotype strains is weak. The immune effect of existing vaccines is not ideal. Therefore, the development of a mixed vaccine for porcine atrophic rhinitis has important guiding significance. The inventors have designed and prepared an inactivated porcine atrophic rhinitis vaccine with the following advantages:

[0030] First, the porcine atrophic rhinitis inactivated vaccine designed and prepared by the present invention contains four antigens, including porcine toxigenic Pasteurella multocida (ZXT + The invention also provides an inactivated porcine atrophic rhinitis vaccine comprising an inactivated antigen of porcine Pasteurella multocida (Pm), an inactivated antigen of porcine Bordetella bronchiseptica (AHBb), and antigens of the N-terminal and C-terminal proteins of the PMT toxin of toxin-producing Pasteurella multocida (rPMT-N and rPMT-C). These antigens not only effectively prevent infection by porcine toxin-producing Pasteurella multocida and Bordetella bronchiseptica, but also effectively prevent nasal turbinate atrophy caused by osteolytics produced by porcine toxin-producing Pasteurella multocida even after infection. Therefore, the porcine atrophic rhinitis inactivated vaccine of the invention can more comprehensively and effectively treat and prevent porcine atrophic rhinitis.

[0031] Secondly, the present invention utilizes the method of expressing the N-terminal and C-terminal proteins of the toxin-producing Pasteurella multocida PMT toxin (rPMT-N and rPMT-C) to replace the traditional inactivated PMT toxin. The rPMT-N and rPMT-C are non-toxic, highly soluble, expressed in large amounts, have a short production time, are simple to manufacture, are highly safe, and have high immunogenicity. Therefore, the porcine atrophic rhinitis inactivated vaccine of the present invention contains more effective and non-toxic rPMT-N and rPMT-C antigens to enhance the immune and preventive effects.

[0032] Finally, the inventors of the present invention have developed an inactivated porcine atrophic rhinitis vaccine with a rationally proportioned combination of four antigens through repeated experiments. This solves the problem of mutual interference between the four antigens and improves the vaccine's immunogenicity. Furthermore, the inventors have also provided a carbomer water adjuvant, which alleviates the problems of traditional adjuvants such as incomplete absorption after injection, low immune efficacy, and significant side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is ZXT + Pm-PCR test result diagram;

[0034] FIG2 is a diagram showing the AHBb-PCR detection results;

[0035] Figure 3 is a diagram showing double enzyme digestion of the recombinant plasmid;

[0036] FIG4 is a diagram showing the identification of rPMT-N and rPMT-C proteins. DETAILED DESCRIPTION

[0037] Please refer to Figures 1-4. The present invention provides a technical solution:

[0038] Preparation of a porcine atrophic rhinitis inactivated vaccine composition, the porcine atrophic rhinitis inactivated vaccine composition comprises porcine toxigenic Pasteurella multocida (ZXT + Pm), porcine bronchiseptica (AHBb), and toxigenic Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) with carbomer water adjuvant;

[0039] The specific steps of the preparation method of the porcine atrophic rhinitis inactivated vaccine composition are as follows:

[0040] Step 1: Store the porcine toxigenic Pasteurella multocida ZXT + The freeze-dried bacteria of strain Pm were inoculated into solid culture medium for resuscitation culture, and single clones were picked and cultured in liquid culture medium. After that, they were expanded in appropriate culture medium for 8-10 hours, and the bacterial liquid was harvested and inactivated.

[0041] Step 2: inoculating the preserved freeze-dried bacteria of Bordetella bronchiseptica AHBb strain into a solid culture medium for recovery culture, picking a single clone and culturing it in a liquid culture medium, then expanding the culture in a suitable culture medium for 10-12 hours, harvesting the bacterial solution, and inactivating it;

[0042] Step 3: The preserved engineered bacteria Rosetta-rPMT-N and Rosetta-rPMT-C freeze-dried bacteria were inoculated into solid culture medium for recovery culture, single clones were picked and cultured in liquid culture medium, and then expanded and induced for expression in a suitable culture medium. The bacteria were harvested by centrifugation and crushed under high pressure, and the target protein was harvested and purified. The N-terminal protein and C-terminal protein of toxin-producing Pasteurella multocida (rPMT-N and rPMT-C proteins) were harvested, sterilized, and the protein concentration was tested;

[0043] Step 4: Inactivate the porcine toxigenic Pasteurella multocida ZXT + The Pm strain, porcine bronchiseptica Pasteurella multocida AHBb strain, and toxigenic Pasteurella multocida N-terminal protein and C-terminal protein are mixed in a certain proportion, added with carbomer water adjuvant, and emulsified to prepare a polyvalent inactivated vaccine.

[0044] The preparation method of the polyvalent inactivated vaccine for porcine atrophic rhinitis of the present invention comprises the following steps: + Pm) content is 1×10 9 CFU / ml, the content of porcine bronchiseptica Bordetella (AHBb) was 1×10 9 CFU / ml, the contents of the N-terminal and C-terminal proteins of the toxin-producing Pasteurella multocida PMT toxin (rPMT-N and rPMT-C) are both 10-80 μg / ml, and are mixed with a certain proportion of carbomer water adjuvant.

[0045] The porcine toxigenic Pasteurella multocida (ZXT + Pm) and porcine bronchiseptica (AHBb) were optimized and cultured until they entered the stationary phase, inactivated with formaldehyde, and treated with Triton X-114 to remove endotoxin. The porcine toxigenic Pasteurella multocida ZXT + The Pm strain is deposited with the number XXXX, the porcine bronchiseptica Boswellia spp. AHBb strain is deposited with the number XXXX, the recombinant expression strain Rosetta-rPMT-N is deposited with the number XXXX, the recombinant expression strain Rosetta-rPMT-C is deposited with the number XXXX, and the porcine toxigenic Pasteurella multocida (ZXT + The cells were harvested when the optimized culture time for Pm) was about 8-10 hours and the optimized culture time for porcine bronchiseptica Bordetella suis (AHBb) was 10-12 hours.

[0046] The toxin-producing Pasteurella multocida PMT toxin N-terminal protein (rPMT-N) is 1461 bases from N1 to 1461, with a protein size of 88 kDa. The C-terminal protein (rPMT-C) is 900 bases from C2959 to 3858, with a protein size of 67 kDa. The proteins were cloned, expressed, and purified using Rosetta-rPMT-N and Rosetta-rPMT-C strains.

[0047] The process of cloning, expression and protein purification of the Rosetta-rPMT-N and Rosetta-rPMT-C strains is as follows:

[0048] (1) Amplifying the gene fragments shown in SEQ.NO.1 and SEQ.NO.2, ligating them into the linear vector pET32a to obtain pET32a-PMT-N and pET32a-PMT-C plasmids, and transforming the pET32a-PMT-N and pET32a-PMT-C plasmids into Escherichia coli Rosetta to obtain recombinant bacteria;

[0049] (2) Cultivating the recombinant bacteria obtained in step (1), then adding 1 mM IPTG to the culture medium, shaking at 30°C for 4 hours, and centrifuging to obtain bacterial cells;

[0050] (3) resuspending the microorganisms described in step (2) in PBS buffer, crushing them under high pressure, and collecting the supernatant by centrifugation to obtain a crude protein solution;

[0051] (4) Purifying the crude protein obtained in step (3) using a Ni column and eluting it with a certain concentration of imidazole for purification;

[0052] (5) After removing imidazole from the purified solution obtained in step (4), the concentration of the purified protein solution (rPMT-N or rPMT-C) was detected using a BCA kit.

[0053] The porcine toxigenic Pasteurella multocida (ZXT + The contents of Pm) and AHBb were both 1×10 9 CFU / ml, and the content of the toxin-producing Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) is 10-80 μg / ml.

[0054] The adjuvant is carbomer water adjuvant, which is mixed with the antigen component in an appropriate ratio.

[0055] The base sequences of the N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the Pasteurella multocida-producing PMT toxin are SEQ.NO.1 and SEQ.NO.2, and the amino acid sequences are SEQ.NO.3 and SEQ.NO.4.

[0056] The porcine toxigenic Pasteurella multocida (ZXT + Pm) and porcine bronchiseptica Bordetella suis (AHBb) content was 1×10 9 CFU / ml, the content of the toxin-producing Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) is 10-80 μg / ml, and is mixed with a certain proportion of carbomer water adjuvant.

[0057] Inactivated porcine toxigenic Pasteurella multocida (ZXT + Pm), porcine bronchiseptica (AHBb) were inactivated by formaldehyde to obtain inactivated antigens, and the inactivated porcine toxigenic Pasteurella multocida (ZXT + Pm) and porcine bronchiseptica (AHBb) were treated with Triton X-114 to remove endotoxins; the N-terminal protein antigen of the PMT toxin of toxin-producing Pasteurella multocida (rPMT-N) is N1 to 1461 protein, 1461 bases, and the protein size is 88kDa, and the C-terminal protein (rPMT-C) is C2959-3858 segment including 900 bases, and the protein size is 67kDa; porcine toxin-producing Pasteurella multocida ZXT + The content of Pm strain is 1×10 9 CFU / ml; the content of porcine bronchiseptica Bordetella AHBb strain is 1×10 9 CFU / ml; the content of the N-terminal and C-terminal proteins of the toxin-producing Pasteurella multocida PMT toxin (rPMT-N and rPMT-C) is 10-80 μg / ml; the veterinary acceptable adjuvant is carbomer water adjuvant.

[0058] A method for using the porcine atrophic rhinitis inactivated vaccine composition according to any of claims 1 to 7, and use of the porcine atrophic rhinitis inactivated vaccine composition in the preparation of a vaccine or medicine for preventing or treating progressive or non-progressive porcine atrophic rhinitis.

[0059] Example 1 Preparation of porcine atrophic rhinitis inactivated vaccine

[0060] 1. Source and identification of the strains of the present invention

[0061] 1.1: Source of strains

[0062] Porcine toxigenic Pasteurella multocida ZXT +The Pm strain (Accession No. XXXX) and the Bordetella bronchiseptica AHBb strain (Accession No. XXXX) were isolated, identified, stored, and supplied by the Key Laboratory of Bacteriology, Ministry of Agriculture, Nanjing Agricultural University. The recombinant expression strain Rosetta-rPMT-N (Accession No. XXXX) and the recombinant expression strain Rosetta-rPMT-C (Accession No. XXXX) were constructed, identified, stored, and supplied by the Key Laboratory of Bacteriology, Ministry of Agriculture, Nanjing Agricultural University.

[0063] 1.2: Identification of bacterial species

[0064] 1.2.1: Porcine toxigenic Pasteurella multocida type D ZXT + Pm strain

[0065] 1.2.1.1: Morphological and biochemical characteristics of porcine toxigenic Pasteurella multocida type D ZXT + The Pm strain is a Gram-negative coccidulide. Under the microscope, the bacteria show typical bipolar staining. Its biochemical characteristics are that it ferments glucose, sucrose, and mannitol, produces acid but not gas, does not ferment maltose, lactose, and rhamnose, and is positive in indole and nitrate reduction tests.

[0066] 1.2.1.2: Cultural characteristics: The bacterium does not grow on MacConkey agar; on blood agar plates, it forms small, water-drop-like colonies with smooth edges, no metallic luster, and no visible hemolysis; it grows well on TSA plates, forming small, transparent, smooth, and moist colonies that exhibit blue fluorescence.

[0067] 1.2.1.3: Molecular biological characteristics, design of gene-specific primers for kmt gene of porcine D-toxigenic Pasteurella multocida, and use of porcine D-toxigenic Pasteurella multocida ZXT + DNA templates were extracted from pure cultures of the Pm strain and subjected to PCR reactions. After agarose gel electrophoresis, the product should be observed under UV transflectometer to yield a specific amplified fragment of 457 bp in length (see Figure 1). Based on the dcbF gene of D-type Pasteurella multocida and the toxA gene of toxigenic P. multocida, specific primers were designed to identify D-type Pasteurella multocida and toxigenic P. multocida, respectively. PCR amplification was performed, and the products were observed under UV light on agarose gel electrophoresis, yielding specific fragments of 647 bp and 864 bp, respectively (see Figure 1).

[0068] 1.2.2: Bordetella bronchiseptica AHBb strain

[0069] 1.2.2.1: Morphological and biochemical characteristics: It is a Gram-negative small coccobacillus. Its biochemical characteristics are that it does not ferment sugars and mannitol. The indole, methyl red and VP tests are negative, the citrate utilization test and nitrate reduction test are positive, and the urease, peroxidase, adenylate cyclase and catalase tests are positive. It does not hydrolyze gelatin, which is consistent with the biochemical characteristics of Bordetella bronchiseptica.

[0070] 1.2.2.2: Culture Characteristics: After incubation on MacConkey agar at 37°C for 40-48 hours, colonies will be slightly yellow. On TSA plates containing 2% fetal bovine serum and 1% coenzyme C for 36 hours at 37°C, colonies will be approximately 0.5 mm in diameter, smooth, translucent, moist, and have neat edges. Phase I bacteria will be observed on Boehringer's medium at 37°C for 40-48 hours, with a β-hemolytic ring.

[0071] 1.2.2.3: Molecular biological characteristics: Gene-specific primers were designed for the fla gene of Bordetella bronchiseptica. PCR reactions were performed using DNA template extracted from a pure culture of the porcine Bordetella bronchiseptica AHBb strain. After agarose gel electrophoresis, a specific amplified fragment of 237 bp in length should be observed under UV transflectance (see Figure 2).

[0072] 1.2.3: PMT genetic engineering expression strains Rosetta-rPMT-N and Rosetta-rPMT-C

[0073] 1.2.3.1: Morphology: It is a Gram-negative bacillus with typical Escherichia coli morphology.

[0074] 1.2.3.2: Culture Characteristics: This bacterium can grow in either Luria-Bert liquid or solid medium (Note 1) containing 100 μg / ml ampicillin. When cultured in solid medium at 37°C for 14–16 hours, round, smooth, raised colonies will form with a milky white sheen.

[0075] 1.2.3.3: Molecular Biological Characterization. pET32a-rPMT-N and pET32a-rPMT-C plasmids were extracted from cultures of the genetically engineered expression strains Rosetta-rPMT-N and Rosetta-rPMT-C, respectively. After double digestion with BamHI and SacI, the digested fragments were identified by 1% agarose gel electrophoresis. pET32a-rPMT-N showed two bands after double digestion, one approximately 1461 bp and the other approximately 5900 bp. pET32a-rPMT-C showed two bands after double digestion, one approximately 900 bp and the other approximately 5900 bp (see Figure 3).

[0076] 1.2.3.4: Protein Expression Characterization: Genetically engineered expression strains Rosetta-rPMT-N and Rosetta-rPMT-C were streaked onto LB plates containing 50 μg / ml ampicillin and incubated at 37°C for 14–16 hours. A single colony of each strain was selected and inoculated into 3 ml of LB liquid medium containing 100 μg / ml ampicillin. The cells were shaken at 37°C at 200 rpm. When the OD600 value reached 0.6–0.8 during the logarithmic growth phase, isopropylthio-β-D-galactopyranoside (IPTG) was added to a final concentration of 0.1 mmol / L and incubated for another 3–4 hours. 1.0 ml of each inducer was centrifuged at 10,000 rpm for 5 minutes. The cells were harvested and resuspended in 100 μl of sterile deionized water. One-quarter volume of 5× SDS-PAGE loading buffer was added. After incubation at 100°C for 10 minutes, 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed. The proteins expressed by the Rosetta-rPMT-N and Rosetta-rPMT-C strains were approximately 88 kDa and 67 kDa, respectively (see Figure 4).

[0077] 2. Preparation of inactivated porcine atrophic rhinitis vaccine

[0078] 2.1: Preparation of bacterial suspension

[0079] 2.1.1: Porcine toxigenic Pasteurella multocida type D ZXT + Preparation of Pm strain bacterial liquid

[0080] Pasteurella multocida ZXT + The freeze-dried bacteria of the Pm strain were streaked onto TSA plates containing 2% newborn calf serum and 1% coenzyme, cultured at 37°C for 24 to 36 hours, and a single typical colony was selected and inoculated into 5 ml of BHI liquid culture medium. The culture was shaken at 37°C and 180 r / min for 10 to 12 hours. The bacterial liquid was then expanded with a 2% inoculum volume and cultured at 37°C and 180 r / min for 10 to 12 hours. Samples were collected for viable bacterial count and purity test.

[0081] 2.1.2: Preparation of Bordetella bronchiseptica AHBb strain

[0082] Lyophilized strains of porcine Bordetella bronchiseptica AHBb were streaked onto TSA plates containing 2% newborn calf serum and 1% coenzyme, cultured at 37°C for 36 to 48 hours, and a single typical colony was selected and inoculated into 5 ml of TSB liquid culture medium. The culture was shaken at 37°C and 180 r / min for 12 to 16 hours. The bacterial liquid was then expanded with a 2% inoculum size and cultured at 37°C and 180 r / min for 12 to 16 hours. Samples were collected for viable bacterial count and purity test.

[0083] 2.2: Inactivation of bacterial solution

[0084] Take the expanded culture of porcine D-type toxigenic Pasteurella multocida ZXT + Slowly add formaldehyde solution to the Pm strain bacterial suspension and the porcine bronchiseptica AHBb strain bacterial suspension to a final concentration of 0.3% (V / V), inactivate at 37°C for 24 to 48 hours, spread the inactivated bacterial suspension on TSA plates, and incubate inverted at 37°C for 24 to 48 hours. Observe and observe for the growth of colonies.

[0085] 2.3: Concentration of bacterial culture

[0086] Take inactivated porcine toxigenic Pasteurella multocida type D ZXT + The bacterial suspension of Pm strain and Bordetella suis AHBb strain was centrifuged to harvest the cells, and the precipitated cells were resuspended in sterile PBS (pH 7.4) buffer and washed three times by centrifugation to prepare a 1×10 11 CFU / ml of bacterial solution.

[0087] 2.4: Preparation of N-terminal and C-terminal proteins of PMT toxin from toxigenic Pasteurella multocida

[0088] 2.4.1: Preparation of bacterial solution

[0089] The lyophilized strains of Rosetta-rPMT-N and Rosetta-rPMT-C were streaked onto LB solid plates containing 100 μg / ml ampicillin and cultured at 37°C for 16 to 18 hours. A single typical colony was then selected and inoculated into LB liquid medium containing 100 μg / ml ampicillin. The culture was shaken at 37°C and 180 rpm for 12 to 16 hours. The bacterial solution was then expanded with a 2% inoculum size and induced for expression. The bacterial solution was harvested and concentrated by centrifugation.

[0090] 2.4.2: Disruption, inactivation and purification of bacterial liquid

[0091] The concentrated bacterial solution was crushed under high pressure (1300Pa), and the crushed bacterial solution was inactivated by adding formaldehyde with a final concentration of 0.3% for 24 hours. The inactivated solution was purified by nickel column, and rPMT-N and rPMT-C proteins were harvested by elution with 75mM imidazole.

[0092] 2.4.3: Protein quantification

[0093] The rPMT-N and rPMT-C proteins harvested after purification were sterile filtered and quantitatively analyzed by BCA assay and Coomassie Brilliant Blue method (SDS-PAGE) to determine the concentrations of rPMT-N and rPMT-C proteins.

[0094] 2.5: Preparation of inactivated porcine atrophic rhinitis vaccine

[0095] Take porcine D-toxigenic Pasteurella multocida ZXT + The inactivated bacterial liquid of Pm strain, the inactivated bacterial liquid of porcine bronchiseptica Bordetella AHBb strain, and the purified toxin-producing Pasteurella multocida PMT toxin N-terminal and C-terminal proteins are mixed in a certain proportion, and a certain amount of carbomer water adjuvant is added thereto. The mixture is shaken at room temperature for 1 hour, and the pH value is adjusted to 7.6-7.8. The finished vaccine is inspected, packaged, and labeled. The inactivated porcine toxin-producing Pasteurella multocida (ZXT + Pm) content is 1×10 9 CFU / ml, the content of porcine bronchiseptica Bordetella (AHBb) was 1×10 9 CFU / ml, and the content of the N-terminal and C-terminal proteins of the toxin-producing Pasteurella multocida PMT toxin (rPMT-N and rPMT-C) was 40 μg / ml.

[0096] Example 2 Preparation of porcine atrophic rhinitis inactivated vaccine 2

[0097] The inactivation of bacterial strains and strains, expression and purification of rPMT-N and rPMT-C were the same as in Example 1.

[0098] The ratio of each antigen in Example 2 is shown in Table 1 below.

[0099] Example 3 Preparation of porcine atrophic rhinitis inactivated vaccine

[0100] The inactivation of bacterial strains and strains, expression and purification of rPMT-N and rPMT-C were the same as in Example 1.

[0101] The ratio of each antigen in Example 3 is shown in Table 1 below.

[0102] Example 4 Preparation of porcine atrophic rhinitis inactivated vaccine

[0103] The inactivation of bacterial strains and strains, expression and purification of rPMT-N and rPMT-C were the same as in Example 1.

[0104] The ratios of the various antigens in Example 4 are shown in Table 1 below.

[0105] Table 1 Ingredient ratio of porcine atrophic rhinitis inactivated vaccine

[0106] Example 1 Inspection of Finished Porcine Atrophic Rhinitis Inactivated Vaccine

[0107] Three batches of vaccine were prepared according to the method of Example 1, namely 202001, 202002 and 202003.

[0108] 1. Vaccine testing

[0109] 1.1 Finished product inspection

[0110] Physical properties, sterility test, filling test and formaldehyde residue determination are carried out in accordance with the appendix of the Chinese Veterinary Pharmacopoeia.

[0111] 1.2 Safety Inspection

[0112] Twenty healthy, susceptible 14-day-old piglets (negative for Bordetella bronchiseptica antibodies in agglutination assays (see Note 1); negative for porcine type D toxigenic Pasteurella multocida PMT blocking ELISA antibodies (see Note 2); and negative for Bordetella bronchiseptica and Pasteurella multocida antigens in nasal swab PCR assays (see Note 3)) were divided into four groups of five pigs each. Groups 1 to 3 served as safety test groups, receiving a 4.0 ml injection of the vaccine into the neck muscle. Group 4 served as the negative control group, receiving a 4.0 ml injection of sterile saline into the neck muscle. The pigs were observed for 14 days after the test.

[0113] 1.3 Effectiveness test

[0114] Twenty healthy susceptible piglets at 14 days of age (negative for antibodies to porcine bronchiseptica Bordetella agglutination test, see Note 1; negative for antibodies to porcine type D toxigenic Pasteurella multocida PMT blocking ELISA, see Note 2; negative for antigens to porcine bronchiseptica Bordetella and Pasteurella multocida PCR test in nasal swabs, see Note 3) were divided into four groups of five pigs each. Groups 1 to 3 were immunized groups, with 1 ml of vaccine injected intramuscularly into the neck of each piglet. 14 days later, the second vaccination was performed with the same dose and method; Group 4 served as the control group and was not vaccinated. 28 days after the second vaccination, all pigs were challenged with the virus, and 0.5 ml of the vaccine containing 8.2×10 6 CFU / ml of Bordetella bronchiseptica; toxigenic Pasteurella multocida was challenged 28 days after the second vaccination. The challenge method was the same as above. Each pig was slowly instilled with 0.5 ml of 1×10 9 CFU / ml of toxigenic Pasteurella multocida was used for 4 consecutive days and observed daily after the infection. All experimental pigs were killed on the 14th day, their noses were transected, and the nasal lesions of the nasal concha were examined and scored and analyzed. The necropsy was performed to observe whether there were any lesions.

[0115] Scoring criteria for porcine nasal turbinates: The pigs were sacrificed, placed in a recumbent position, and their heads slightly elevated. A transverse incision was made across the snout between the first and second molars. The cross-section was then examined for nasal turbinates atrophy (TA) and nasal septum deviation (NSD). Each turbinate atrophy was scored on a scale of 0 to 4 (0: normal, no lesions; 1: very small atrophy, with nearly half of one turbinate atrophied; 2: mild atrophy, with more than half of one turbinate atrophied; 3: moderate atrophy, with ankylosing of the turbinate; 4: severe atrophy, with the entire turbinate atrophy absent). Nasal septum deviation was scored on a scale of 0 to 2 (0: normal; 1: mild deviation; 2: severe deviation). Each pig's turbinate atrophy score was calculated by adding the four scores for turbinate atrophy and septum deviation, for a maximum score of 18.

[0116] 2. Laboratory test results

[0117] 2.1 Physical properties inspection

[0118] Random samples were taken from three batches of vaccine for characterization. This product is a translucent suspension with a small amount of sediment at the bottom after prolonged storage. After shaking, it becomes a homogeneous suspension (see Table 2).

[0119] Table 2 Test results of properties of 3 batches of laboratory finished products

[0120] 2.2 Sterility test

[0121] Five bottles of vaccine were randomly selected from each of the three batches and inoculated into TG medium. After culturing at 37°C for 3 days, they were inoculated into thioglycollate medium and casein medium slant, and incubated at 37°C and 25°C, respectively. After culturing for 7 days, all (5 / 5) showed sterile growth (see Table 3), meeting the requirements.

[0122] Table 3 Sterility test results of each batch of vaccine

[0123] 2.3 Filling quantity inspection

[0124] Five bottles of vaccine were randomly sampled from each of the three batches for volume inspection, and all results met the requirements (Table 6). The volume of the five bottles sampled from batch 1601 was 20.5, 21.2, 20.8, 20.6, and 21.1 mL, respectively; the volume of the five bottles sampled from batch 1602 was 20.8, 21.5, 20.6, 20.5, and 20.9 mL, respectively; and the volume of the five bottles sampled from batch 1603 was 20.4, 20.6, 20.9, 20.7, and 21.1 mL, respectively (see Table 4), all meeting the requirements.

[0125] Table 4 Inspection results of vaccine loading in each batch

[0126] 2.4 Formaldehyde Residue Inspection

[0127] Five bottles of vaccine were randomly sampled from each of the three batches for formaldehyde residue testing. The results (Table 8) showed that the formaldehyde residues in the 201601, 201602, and 201603 batches were 0.11%, 0.10%, and 0.13%, respectively (see Table 5), meeting the requirements.

[0128] Table 5 Determination of formaldehyde residues in each batch of vaccine

[0129] 2.5 Safety Check

[0130] The three batches of vaccine were injected into the neck muscles of 14-day-old healthy susceptible piglets with 4.0 ml of the vaccine. After 14 days of observation, no local or systemic adverse reactions due to the vaccine were observed (see Table 6).

[0131] Table 6 Safety test results of a single dose of vaccination in 14-day-old piglets

[0132] 2.6 Effectiveness test

[0133] After the challenge, a few pigs in the non-immune challenge control group and the three vaccine-immunized groups experienced elevated body temperatures for 1-2 days, while the majority of the other pigs showed no significant clinical abnormalities. During observation for 14 days after the challenge, the relative daily weight gain of the immunized pigs was not significantly different from that of the blank control group (P>0.05), but was significantly higher than that of the non-immune challenge control group (P<0.05). The nasal turbinates of the immunized pigs showed slight curling and atrophy, which was not significantly different from the blank control group. However, the nasal turbinates of the challenge control group were severely atrophied and the lung lesions were obvious, which were significantly different from the blank control group (P<0.05) (see Table 7).

[0134] Table 7. Statistical results of clinical symptoms of three batches of vaccine-immunized pigs after challenge (I)

[0135] Table 7. Statistical results of clinical symptoms of three batches of vaccine-immunized pigs after challenge (II)

[0136] Tables 2 to 7 show that the physical properties, sterility test, safety test and efficacy test of three batches of porcine atrophic rhinitis inactivated vaccine were met, and all of them met the requirements. They had good protection against the attacks of porcine bronchiseptica Bordetella and toxigenic Pasteurella multocida. The vaccine prepared according to Example 1 met our design.

[0137] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the appended claims rather than the above description. Therefore, it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any figure signs in the claims should not be regarded as limiting the claims involved.

[0138] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Preparation of a porcine atrophic rhinitis inactivated vaccine composition, characterized in that: The porcine atrophic rhinitis inactivated vaccine composition comprises porcine toxin-producing Pasteurella multocida (ZXT + Pm), porcine Bordetella bronchiseptica (AHBb), and toxigenic Pasteurella multocida PMT toxin N-terminal and C-terminal proteins (rPMT-N and rPMT-C) with carbomer water adjuvant; The specific steps of the preparation method of the porcine atrophic rhinitis inactivated vaccine composition are as follows: Step 1: Store the porcine toxigenic Pasteurella multocida ZXT + The freeze-dried bacteria of strain Pm were inoculated into solid culture medium for resuscitation culture, and single clones were selected for culture in liquid culture medium, and then expanded for 8-10 hours in appropriate culture medium, and the bacterial liquid was harvested and inactivated; Step 2: inoculating the preserved lyophilized bacteria of Bordetella bronchiseptica AHBb strain into a solid culture medium for recovery culture, picking a single clone colony for culture in a liquid culture medium, and then expanding the culture in a suitable culture medium for 10-12 hours, harvesting the bacterial solution, and inactivating it; Step 3: Inoculate the preserved engineered bacteria Rosetta-rPMT-N and Rosetta-rPMT-C freeze-dried bacteria into solid culture medium for recovery culture, pick monoclonal colonies for culture in liquid culture medium, then expand culture in suitable culture medium and induce expression, harvest the bacteria by centrifugation for high-pressure crushing, harvest the target protein and purify it, harvest the N-terminal protein and C-terminal protein (rPMT-N and rPMT-C proteins) of toxin-producing Pasteurella multocida, treat them aseptically and detect the protein concentration; Step 4: Inactivate the porcine toxin-producing Pasteurella multocida ZXT + The Pm strain, the porcine bronchiseptica Boerseniae AHBb strain, the N-terminal protein and the C-terminal protein of toxin-producing Pasteurella multocida are mixed in a certain proportion, and a carbomer water adjuvant is added and the mixture is emulsified to prepare a polyvalent inactivated vaccine.

2. The preparation of a porcine atrophic rhinitis inactivated vaccine composition according to claim 1, characterized in that: The porcine toxigenic Pasteurella multocida (ZXT + Pm) and porcine bronchiseptica Bordetella (AHBb) were optimized and cultured until they entered the stationary phase, inactivated with formaldehyde, and treated with Triton X-114 to remove endotoxin.

3. The preparation of a porcine atrophic rhinitis inactivated vaccine composition according to claim 1, characterized in that: The toxin-producing Pasteurella multocida PMT toxin N-terminal protein (rPMT-N) is N1-1461, a total of 1461 bases, and the protein size is 88kDa. The C-terminal protein (rPMT-C) is C2959-3858, including 900 bases, and the protein size is 67kDa. It is cloned, expressed and purified by Rosetta-rPMT-N and Rosetta-rPMT-C strains. The process of cloning, expression and protein purification of the Rosetta-rPMT-N and Rosetta-rPMT-C strains is as follows: (1) Amplifying the gene fragments shown in SEQ.NO.1 and SEQ.NO.2, connecting them to the linear vector pET32a to obtain pET32a-PMT-N and pET32a-PMT-C plasmids, and transforming the pET32a-PMT-N and pET32a-PMT-C plasmids into Escherichia coli Rosetta to obtain recombinant bacteria; (2) culturing the recombinant bacteria obtained in step (1), then adding 1 mM IPTG to the culture solution, shaking at 30° C. for 4 h, and centrifuging to obtain bacterial cells; (3) resuspending the cells obtained in step (2) in PBS buffer, crushing them under high pressure, and collecting the supernatant by centrifugation to obtain a crude protein solution; (4) Purifying the crude protein obtained in step (3) using a Ni column and eluting it with a certain concentration of imidazole for purification; (5) After removing imidazole from the purified solution obtained in step (4), the concentration of the purified protein solution (rPMT-N or rPMT-C) was detected using a BCA kit.

4. The preparation of a porcine atrophic rhinitis inactivated vaccine composition according to claim 1, characterized in that: The porcine toxigenic Pasteurella multocida (ZXT + The contents of Pm) and AHBb were both 1×10 9 CFU / ml, and the content of the N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the toxin-producing Pasteurella multocida PMT toxin is 10-80 μg / ml.

5. The preparation of a porcine atrophic rhinitis inactivated vaccine composition according to claim 1, characterized in that: The adjuvant is carbomer water adjuvant, which is mixed with the antigen component in an appropriate ratio.

6. The preparation of a porcine atrophic rhinitis inactivated vaccine composition according to claim 1, characterized in that: The base sequences of the N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the PMT toxin produced by Pasteurella multocida are SEQ.NO.1 and SEQ.NO.2, and the amino acid sequences are SEQ.NO.3 and SEQ.NO.

4.

7. The preparation of a porcine atrophic rhinitis inactivated vaccine composition according to claim 1, characterized in that: The porcine toxigenic Pasteurella multocida (ZXT + Pm) and AHBb (AHBb) content was 1×10 9 CFU / ml, the content of the N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the toxin-producing Pasteurella multocida PMT toxin is 10-80 μg / ml, and is mixed with a certain proportion of carbomer water adjuvant.

8. A method for using the porcine atrophic rhinitis inactivated vaccine composition according to any of claims 1 to 7, characterized in that: The invention relates to an application of the porcine atrophic rhinitis inactivated vaccine composition in preparing a vaccine or medicine for preventing or treating progressive or non-progressive porcine atrophic rhinitis.

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