CN114146171B - Swine atrophic rhinitis inactivated vaccine and preparation method thereof - Google Patents
Swine atrophic rhinitis inactivated vaccine and preparation method thereof Download PDFInfo
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- CN114146171B CN114146171B CN202111427009.7A CN202111427009A CN114146171B CN 114146171 B CN114146171 B CN 114146171B CN 202111427009 A CN202111427009 A CN 202111427009A CN 114146171 B CN114146171 B CN 114146171B
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- pasteurella multocida
- toxigenic
- swine
- atrophic rhinitis
- pigs
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Abstract
The invention discloses an inactivated vaccine for atrophic rhinitis of pigs and a preparation method thereof, wherein the vaccine is prepared by mixing toxigenic pasteurella multocida, bordetella bronchiseptica, toxigenic pasteurella multocida PMT toxin N-terminal and C-terminal proteins with carbomer water adjuvant in a certain proportion, has good immunogenicity, can effectively prevent progressive and non-progressive atrophic rhinitis of pigs, and improves the productivity of pigs. The inactivated vaccine for the atrophic rhinitis of the pigs, which is prepared by the invention, contains antigen components of ZXT+Pm and AHBb, and rPMT-N and rPMT-C are added, so that the inactivated vaccine not only prevents the atrophic rhinitis of the pigs caused by ZXT+Pm and AHBb, but also avoids the osteolytic effect generated by toxin proteins secreted by ZXT+Pm, and strengthens the immunoprotection effect on the atrophic rhinitis of the pigs.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an inactivated vaccine for atrophic rhinitis of pigs and a preparation method thereof.
Background
Atrophic rhinitis (Progressive atrophic rhinitis, PAR) is a chronic respiratory disease, is mainly characterized by extensive nasal expansion and atrophy of mucous membrane, submucosa and bone tissue, has high contact infectivity, is also called infectious atrophic rhinitis or chronic atrophic rhinitis, and is one of the important infectious diseases recognized as serious harm to the production and development of intensive pig farms. PAR is mainly caused by infection of toxigenic Pasteurella multocida and B.bronchiseptica, and often secondary infection with other respiratory diseases, such as mycoplasma hyopneumoniae, blue ear disease, wheezing disease, etc. The disease has wide epidemic range and is commonly generated all over the world, the incidence rate is relatively high in developed areas of pig industry, the pathogen detection rate of pig groups is high, and the disease is one of main diseases of a hazard-standardized pig farm and is listed as B-type diseases by animal health organizations in the world. Therefore, the development of more effective and safer inactivated vaccine for atrophic rhinitis of pigs has become an urgent need.
The prior atrophic rhinitis of pigs is divided into progressive atrophic rhinitis and non-progressive atrophic rhinitis, the progressive atrophic rhinitis (Progressive atrophic rhinitis, PAR) is mainly caused by the toxigenic strain (DNT+Pm) of Pasteurella multocida (Pasteurella multoeida, pm) (most of which are D-type, a few of which are A-type); another type of Non-progressive atrophic rhinitis (Non-progressive atrophie rhinitis, NPAR) is mainly caused by phase I bacteria of B.bronchiseptica (Bordetella bronchiseptica, bb). The latter is lighter when infected alone and heavier when both are mixed or infected secondarily.
The pathogenic agent of PAR is bordetella bronchiseptica (Bb) and toxigenic Pasteurella multocida (Pm). Bb gram staining is negative, short bacillus without spore has periflagellum and can move; the capsule is mainly dyed in two poles, is arranged in pairs or scattered ways and can be divided into 3 bacterial phases of phase I bacteria, phase II bacteria and phase III bacteria, but the pathogenic effect is that the phase I bacteria are typical, the phase I bacteria are pearl-shaped or semicircular, the diameter is less than 1mm, the white, smooth and compact, and the boundary of a beta hemolysis ring is not clear. The bacterial colony can be kept in phase I bacteria by adding 5% -10% of sterile defibrinated sheep whole blood into abalone-ginger culture medium (BG). Clinical studies prove that single bordetella bronchiseptica infection is not serious in onset, and the defect of nasal bone is renewable, and belongs to non-progressive atrophic rhinitis. Pasteurella multocida (Pm) belongs to gram-negative bacteria, and is spherical or short rod-shaped with rounded ends, and the same as Bb is that spores cannot be formed, except that Pm cannot move. Based on the lipopolysaccharide antigen classification, pm strains can be divided into 5 capsular serotypes (a, B, D, E and F) and 16 individual cell serotypes (1 to 16), with serotypes a and D being one of the pathogens responsible for progressive swine atrophic rhinitis and pasteurella multocida type a and type D producing exotoxins. Studies have shown that: pasteurella multocida has a variety of virulence factors including toxins, adhesins and pili, especially the greatest hazard of the skin necrosis toxin PMT, a protein of about 146 kDa.
Symptoms after the atrophic rhinitis infection of pigs appear in pigs with 3 to 4 weeks of age or weaned pigs, and the age of onset of the pigs is inversely related to the occurrence of diseases. The sick pigs show the characteristics of catarrhal rhinitis with different degrees, frequently sneeze, unsmooth breathing, snoring and the like; piglets show appetite reduction, and the nostrils are continuously serous or mucopurulent secretion, but the piglets can recover to be normal when the illness state is lighter. In the later stage of infection, the turbinates and the girder bones of the sick pigs start to shrink and deform under the stimulation of inflammation, the turbinates lesions extend from the abdomen side to the back side, the nostrils at two sides are different in size, and the nose ends are inclined towards the sick side, so that the turbinates are caused.
With the research on the pathogenic mechanism of atrophic rhinitis of pigs, the best method for preventing the disease is vaccine immunization. Commercial vaccines in the market at present mainly comprise inactivated vaccine and toxoid vaccine, and the immune effect of the toxoid vaccine is superior to that of the inactivated vaccine, but the ideal effect cannot be achieved. Therefore, the inactivated vaccine (ZXT+Pm and AHBb) and the recombinant pasteurellosis toxin proteins ((rPMT-N and rPMT-C)) are adopted in the study, so that on one hand, piglets can be protected from the attack of Bb and Pm, and on the other hand, the osteolytic effect of toxin proteins generated by T+Pm by the piglets is avoided, and thus, the occurrence of atrophic rhinitis of the piglets is prevented and protected more comprehensively.
Disclosure of Invention
Aiming at the defects of the existing vaccine, the invention provides an inactivated vaccine for atrophic rhinitis of pigs and a preparation method thereof, which are used for solving the technical problem that related vaccines in the prior art have poor universality for different strains and toxins produced.
The swine toxigenic pasteurella multocida ZXT+Pm strain has been preserved, the preservation unit: china center for type culture Collection, address: chinese, wuhan, university of Wuhan, date of preservation: 2021, 11, 15, deposit number: cctccc No: m20211427 classification nomenclature: pasteurella multoeida ZXT-Pm.
The porcine bordetella bronchiseptica AHBb strain has been preserved, and the preservation unit is: china center for type culture Collection, address: chinese, wuhan, university of Wuhan, date of preservation: 2021, 11, 08, deposit number: cctccc No: m20211379 classification nomenclature: boretella bronchischiica ahbb.
The recombinant expression bacterium Rosetta-rPMT-N of the invention is preserved, and the preservation unit is: china center for type culture Collection, address: chinese, wuhan, university of Wuhan, date of preservation: 2021, 11, 05, storage number: cctccc No: m20211371 classification nomenclature: escherichia coli Rosetta-rPMT-N.
The recombinant expression bacterium Rosetta-rPMT-C has been preserved, and the preservation unit is: china center for type culture Collection, address: chinese, wuhan, university of Wuhan, date of preservation: 2021, 11, 05, storage number: cctccc No: m20211372 classification nomenclature: escherichia coli Rosetta-rPMT-C.
The swine toxigenic pasteurella multocida (ZXT+Pm) is harvested when the optimized culture is about 8-10 hours and the optimized culture is about 10-12 hours.
The inactivated swine toxigenic Pasteurella multocida (ZXT+Pm) and the swine bordetella bronchiseptica (AHBb) are all inactivated by formaldehyde to obtain an inactivated antigen.
The inactivated swine toxinogenic Pasteurella multocida (ZXT+Pm) and porcine bordetella bronchiseptica (AHBb) of the present invention are treated with Triton X-114 to remove endotoxins.
The N-terminal antigen (rPMT-N) protein of the toxin of the toxigenic pasteurella multocida is N1-1461 protein, 1461 base, the protein size is 88kDa, the C-terminal protein (rPMT-C) of the toxin of the toxigenic pasteurella multocida is C2959-3858 segment comprising 900 base, and the protein size is 67kDa.
The content of the pasteurella multocida ZXT+Pm strain of the invention is 1 multiplied by 10 9 CFU/ml。
The content of the bordetella bronchiseptica AHBb strain of the invention is 1 multiplied by 10 9 CFU/ml。
The content of N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the toxigenic pasteurellosis bacillus PMT toxin is 10-80 mug/ml.
The veterinary acceptable adjuvant of the invention is carbomer water adjuvant.
The invention also aims to provide a preparation method of the swine atrophic rhinitis inactivated vaccine, which comprises the following steps:
(1) Inoculating the stored freeze-dried strain ZXT+Pm strain of the swine toxigenic pasteurellosis bacillus to a solid culture medium for resuscitating culture, selecting a monoclonal to culture in a liquid culture medium, then performing expansion culture in a proper culture medium for 8-10 hours, harvesting bacterial liquid, and inactivating.
(1) Inoculating the preserved freeze-dried strain of the bordetella bronchiseptica AHBb strain into a solid culture medium for resuscitating culture, picking up a monoclonal strain for culture in a liquid culture medium, then performing expansion culture in a proper culture medium for 10-12 hours, harvesting bacterial liquid, and inactivating.
(3) Inoculating the stored engineering bacteria Rosetta-rPMT-N and Rosetta-rPMT-C freeze-dried bacteria into a solid culture medium for resuscitating culture, selecting a monoclonal to culture in the liquid culture medium, then performing amplification culture in a proper culture medium, performing induced expression, centrifugally harvesting the bacteria, crushing under high pressure, harvesting target proteins, purifying and harvesting N-terminal proteins and C-terminal proteins (rPMT-N and rPMT-C proteins) of the toxigenic Pasteurella multocida, performing aseptic treatment, and detecting the protein concentration.
(4) Mixing the inactivated porcine toxigenic pasteurella multocida ZXT+Pm strain, porcine bordetella bronchiseptica AHBb strain and toxigenic pasteurella multocida N-terminal protein and C-terminal protein according to a certain proportion, adding carbomer water adjuvant, emulsifying and making into the invented multivalent inactivated vaccine.
Preferably, the method for preparing the multivalent inactivated vaccine for the atrophic rhinitis of pigs comprises the step of inactivating the porcine toxigenic pasteurella multocida (ZXT+Pm) with the content of 1 multiplied by 10 9 CFU/ml, content of Brucella bronchiseptica (AHBb) of 1×10 9 CFU/ml, the N-end and C-end proteins (rPMT-N and rPMT-C) of toxigenic Pasteurella multocida PMT toxin are 10-80 mug/ml, and mixed with carbomer water adjuvant according to a certain proportion.
The invention designs and prepares the inactivated vaccine for the atrophic rhinoitis of pigs, which has the advantages that the host of the atrophic rhinitis of pigs is more, the distribution is wide, the serotype distribution in different areas is different, the cross protection capability between the strains of different serotypes is weak, and the immune effect of the existing vaccine is not ideal, so the development of the mixed vaccine for the atrophic rhinitis of pigs has important guiding significance, and the inactivated vaccine for the atrophic rhinoitis of pigs has the following advantages:
firstly, the inactivated vaccine for the atrophic rhinitis of the pigs, which is designed and prepared by the invention, contains four antigens, namely the inactivated antigen of the toxigenic pasteurella multocida (ZXT+Pm) of the pigs, the inactivated antigen of the toxigenic pasteurella multocida (AHBb) of the pigs, and the N-terminal and C-terminal protein (rPMT-N and rPMT-C) antigens of the toxigenic pasteurella PMT of the pigs, so that the infection of the toxigenic pasteurella multocida and the toxigenic pasteurella of the pigs is effectively prevented, and the turbinate atrophy caused by the osteolytic produced by the toxigenic pasteurella multocida of the pigs is effectively prevented even after the infection, so that the inactivated vaccine for the atrophic rhinitis of the pigs can treat and prevent the atrophic rhinitis of the pigs more comprehensively and effectively.
Secondly, the invention uses the N-terminal and C-terminal proteins (rPMT-N and rPMT-C) of the toxin-producing pasteurellosis bacillus PMT toxin to replace the traditional inactivated PMT toxoid method, wherein the rPMT-N and the rPMT-C have the characteristics of non-toxicity, high expression solubility, large expression quantity, short production time, simple process, high safety, high immunogenicity and the like, so that the porcine atrophic rhinitis inactivated vaccine contains better effective and nontoxic rPMT-N and rPMT-C antigens to increase the immune and preventive effects.
Finally, the pig atrophic rhinitis inactivated vaccine of the invention obtains four antigens with reasonable proportion through repeated experiments of the inventor, solves the problem that the four antigens interfere with immune effect mutually, and improves the immunogenicity of vaccine. In addition, the inventor also provides a carbomer water adjuvant, which reduces the problems of incomplete absorption, low immune effect, large side reaction and the like of the traditional adjuvant after injection.
Drawings
FIG. 1 is a diagram showing the results of PCR detection of swine D-type toxigenic Pasteurella multocida of the present invention. Lanes in panel a: m, DL2000 DNA Maker,1, pm,2, negative. Lanes in panel B: m, DL2000 DNA Maker,1, pmD,2, positive. Lanes in panel C: m, DL2000 DNA Maker,1, toxA,2, negative.
FIG. 2 is a diagram showing the results of PCR detection of B.bronchiseptica according to the present invention. Lanes in the figure: m, DL2000 DNA Maker,1, bb,2, bb,3, negative.
FIG. 3 is a diagram showing the identification of recombinant plasmids by double digestion. Lanes in the figure: MDL10kb DNA Marker,2, pET32a-rPMT-N,3, pET32a-rPMT-C.
Detailed Description
The invention is further described below in connection with specific embodiments, which are exemplary only and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the technical solution of the present invention without departing from the spirit and scope of the invention, but these modifications and substitutions fall within the scope of the present invention.
Example 1 preparation of inactivated vaccine against atrophic rhinitis in pigs
1 Source and identification of the species according to the embodiment of the invention
1.1 Source of the Strain
Pig toxigenic pasteurella multocida ZXT+Pm strain collection number CCTCC M20211427
ZXT-Pm, and the collection number CCTCC M20211379 of the porcine bordetella bronchiseptica AHBb strain are separated, identified, stored and supplied by a bacteriological key laboratory of Nanjing university of agriculture. Recombinant expression bacteria Rosetta-rPMT-N preservation number CCTCC M20211371 and recombinant expression bacteria Rosetta-rPMT-C preservation number CCTCC M20211372 are constructed, identified, stored and supplied by the bacteriology key laboratory of Nanjing university of agriculture.
1.2 identification of species
1.2.1 pig D-toxigenic Pasteurella multocida ZXT+Pm strain
1.2.1.1 the strain ZXT+Pm of the pig D-type toxigenic pasteurella multocida with morphological and biochemical characteristics is gram-negative small bacillus, and the strain is typically bipolar-stained under a microscope, and the biochemical characteristics are that glucose, sucrose and mannitol are fermented, acid production is not carried out, gas production is not carried out, maltose, lactose and rhamnose are not fermented, and indole and nitrate reduction tests are positive.
1.2.1.2 culture characteristics the strain did not grow on MAIKAI agar; forming water drop-like microcolonies with smooth edges on blood agar plates, and having no metallic luster and no visible hemolysis; the growth on TSA plate is good, the colony is small, transparent, smooth, moist, it presents blue fluorescence.
1.2.1.3 molecular biological Properties Gene-specific primers were designed for swine D-type toxigenic Pasteurella multocida kmt gene, and PCR was performed using pure culture extract DNA template of swine D-type toxigenic Pasteurella multocida ZXT+Pm strain, and after agarose gel electrophoresis, a specific amplified fragment of 457bp in length was observed under an ultraviolet transilluminator (Panel A in FIG. 1). Specific primers are designed according to the dcbF gene of the type D Pasteurella multocida and the toxigenic Pasteurella multocida toxA gene respectively for identifying the type D Pasteurella multocida and the toxigenic Pasteurella multocida, PCR amplification is carried out, and the products are subjected to agarose gel electrophoresis and observed under an ultraviolet lamp to respectively amplify specific fragments of 647bp (diagram B in figure 1) and 864bp (diagram C in figure 1).
1.2.2 porcine bordetella bronchiseptica AHBb strain
1.2.2.1 the form and biochemical characteristics are gram negative globus bacillus, the biochemical characteristics are non-fermented sugar and mannitol, indole, methyl red and VP tests are negative, citrate utilization tests and nitrate reduction tests are positive, urease, peroxidase, adenylate cyclase and thixotropic tests are positive, gelatin is not hydrolyzed, and the biological characteristics of the porcine bordetella bronchiseptica are met.
1.2.2.2 culture characteristics of the MAIKAI agar culture medium, culturing for 40-48 hours at 37 ℃, wherein colonies are yellowish; the colonies were grown on TSA plates containing 2% fetal bovine serum and 1% coenzyme for 36 hours at 37℃and had a diameter of about 0.5mm, and were smooth, translucent, moist and clean at the edges. The bacterial phase is phase I bacteria, and beta-hemolytic ring is observed after culturing for 40-48 hours at 37 ℃ on Bao Jiangshi culture medium.
1.2.2.3 molecular biological Properties Gene-specific primers were designed for the Brucella bronchiseptica fla gene, and PCR was performed using pure cultures of the Brucella bronchiseptica AHBb strain to extract DNA templates, and after agarose gel electrophoresis, specific amplified fragments with a length of 237bp were observed under an ultraviolet transilluminator, as shown in FIG. 2.
1.2.3PMT genetically engineered expression bacteria Rosetta-rPMT-N and Rosetta-rPMT-C strains
1.2.3.1 the form is gram negative and has a typical E.coli form.
1.2.3.2 culture Properties growth was achieved by culture in LB (Luria-Bert) liquid or solid medium containing 100. Mu.g/ml ampicillin. Culturing in solid culture medium at 37 deg.c for 14-16 hr to obtain circular, smooth, raised and milky glossy colony.
1.2.3.3 molecular biological Properties pET32a-rPMT-N and pET32a-rPMT-C plasmids were extracted with cultures of genetically engineered expression strains Rosetta-rPMT-N and Rosetta-rPMT-C, respectively, and after double digestion with BamHI and SacI, the digested fragments were identified by 1% agarose electrophoresis. After double cleavage, pET32a-rPMT-N has two bands, one has a fragment size of about 1461bp and the other has a fragment size of about 5900bp. After double cleavage, pET32a-rPMT-C has two bands, one with a fragment size of about 900bp and the other with a fragment size of about 5900bp, as shown in FIG. 3.
1.2.3.4 protein expression Properties genetically engineered expression strains Rosetta-rPMT-N and Rosetta-rPMT-C were streaked on LB plates containing 50. Mu.g/ml ampicillin, respectively, and incubated at 37℃for 14-16 hours. Single bacterial colonies of Rosetta-rPMT-N and Rosetta-rPMT-C strains are respectively picked, inoculated into 3ml LB liquid medium containing 100 mug/ml ampicillin, shake cultured by a shaking table at 37 ℃ for 200r/min until the OD600 value in logarithmic growth phase reaches 0.6-0.8, added with isopropyl thiobeta-D-galactoside (IPTG) to a final concentration of 0.1mmol/L, and continuously cultured for 3-4 hours. 1/4 volumes of 5 XSDS-PAGE loading buffer was added to 1/0 ml of inducer, centrifuged at 10000r/min for 5 minutes, the cells were collected, resuspended in 100. Mu.l of sterile deionized water. After 10 minutes of water bath at 100deg.C, 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed, and the proteins expressed by Rosetta-rPMT-N and Rosetta-rPMT-C strains were approximately 88kDa and 67kDa, respectively.
Preparation of 2 pig atrophic rhinitis inactivated vaccine
2.1 preparation of bacterial liquid
2.1.1 preparation of bacterial liquid of swine D-type toxigenic Pasteurella multocida ZXT+Pm strain
The method comprises the steps of streaking and inoculating a freeze-dried strain of porcine D-type toxigenic Pasteurella multocida ZXT+Pm strain to a TSA plate containing 2% of new born calf serum and 1% of coenzyme, culturing at 37 ℃ for 24-36 hours, selecting single typical bacterial colonies, inoculating to 5ml of BHI liquid culture medium, culturing at 37 ℃ for 10-12 hours under shaking at 180r/min, performing expansion culture on bacterial liquid with 2% inoculum size, culturing at 37 ℃ for 10-12 hours under shaking at 180r/min, and harvesting and sampling for viable count and pure detection.
2.1.2 preparation of a bacterial solution of the bordetella bronchiseptica AHBb Strain
The freeze-dried strain of the bordetella bronchiseptica AHBb strain is streaked and inoculated on a TSA plate containing 2% of newborn calf serum and 1% of coenzyme, the culture is carried out for 36 to 48 hours at 37 ℃, single typical colonies are selected, inoculated on 5ml of TSB liquid culture medium, the shake culture is carried out for 12 to 16 hours at 37 ℃ and 180r/min, then the bacterial liquid is subjected to expansion culture with the inoculum size of 2%, the shake culture is carried out for 12 to 16 hours at 37 ℃ and 180r/min, and the living bacterial count and the pure detection are carried out by harvesting and sampling.
2.2 inactivation of bacterial liquids
And (3) taking the strain liquid of the expanded and cultured pig D-type toxigenic pasteurella multocida ZXT+Pm strain and the strain liquid of the pig bronchogenic bordetella AHBb strain, slowly adding formaldehyde solution into the strain liquid to reach the final concentration of 0.3% (V/V), inactivating the strain liquid for 24-48 hours at 37 ℃, coating the inactivated strain liquid on a TSA plate, inversely culturing the strain liquid for 24-48 hours at 37 ℃, and observing the growth of the strain liquid which should be aseptic.
2.3 concentration of bacterial liquid
Collecting inactivated strain of Pasteurella multocida ZXT+Pm strain of pig and strain of Bacillus carborundum AHBb strain of pig, centrifuging to obtain thallus, re-suspending the precipitated thallus with sterile PBS (pH 7.4) buffer solution, centrifuging, washing for 3 times, and making into 1×10 concentration 11 CFU/ml bacterial liquid.
2.4 preparation of N-and C-terminal proteins of toxin-producing Pasteurella multocida PMT toxin
2.4.1 preparation of bacterial liquid
The Rosetta-rPMT-N and Rosetta-rPMT-C freeze-dried strains are streaked and inoculated on an LB solid plate containing 100 mu g/ml ampicillin, cultured for 16-18 hours at 37 ℃, then single typical colonies are selected, inoculated on an LB liquid culture medium containing 100 mu g/ml ampicillin, cultured for 12-16 hours at 37 ℃ under 180r/min shaking, then bacterial liquid is subjected to expansion culture and induced expression with 2% inoculum size, and the bacterial liquid is obtained and concentrated by centrifugation.
2.4.2 crushing, inactivating and purifying the bacterial liquid
And (3) crushing the concentrated bacterial liquid under high pressure (1300 Pa), adding formaldehyde with the final concentration of 0.3% into the crushed bacterial liquid, inactivating for 24 hours, purifying the inactivated liquid by a nickel column, and eluting with 75mM imidazole to obtain rPMT-N and rPMT-C proteins.
2.4.3 protein quantification
The rPMT-N and rPMT-C proteins harvested after purification were subjected to sterile filtration and the rPMT-N and rPMT-C protein concentrations were determined by BCA assay and quantitative analysis by Coomassie Brilliant blue (SDS-PAGE).
2.5 preparation of inactivated vaccine for atrophic rhinitis of pigs
Mixing pig D-type toxigenic pasteurella multocida ZXT+Pm strain inactivated bacterial liquid, pig bronchogenic bordetella multocida AHBb strain inactivated bacterial liquid, purified toxigenic pasteurella multocida PMT toxin N end and C end protein according to a certain proportion, adding a certain amount of carbomer water adjuvant into the mixture, oscillating for 1 hour at room temperature, regulating pH value to 7.6-7.8, checking, split charging and labeling to obtain the finished vaccine, wherein the content of the inactivated pig toxigenic pasteurella multocida (ZXT+Pm) is 1 multiplied by 10 9 CFU/ml, content of Brucella bronchiseptica (AHBb) of 1×10 9 CFU/ml, the N-and C-terminal proteins (rPMT-N and rPMT-C) of the toxin-producing pasteurella multocida PMT toxin were 40. Mu.g/ml.
Example 2 preparation of an inactivated vaccine against atrophic rhinitis in pigs 2
The strain and inactivation of the strain, expression and purification of rPMT-N and rPMT-C were the same as in example 1.
Example 3 preparation of inactivated vaccine against atrophic rhinitis in pigs 3
The strain and inactivation of the strain, expression and purification of rPMT-N and rPMT-C were the same as in example 1.
The ratio of the antigens of example 2 and example 3 is shown in Table 1 below.
Table 1 composition ratio of inactivated vaccine for atrophic rhinitis of pigs
Example 4 inspection of finished porcine atrophic rhinitis inactivated vaccine
1 vaccine batch was prepared according to the methods of example 1, example 2, example 3, respectively, with vaccine batch numbers 202001, 202002, and 202003, respectively.
5 testing of vaccine
1.1 inspection of finished products
And (3) carrying out physical properties, sterility test, loading quantity test and determination of formaldehyde residue according to annex of Chinese animal pharmacopoeia.
1.2 safety inspection
The 14-day-old healthy susceptible piglets (the porcine bordetella bronchiseptica agglutination detection antibody is negative, the porcine D-type toxigenic pasteurella multocida PMT blocking ELISA antibody is negative, and the nasal swab porcine bordetella bronchiseptica and the pasteurella multocida PCR detection antigen are negative) are divided into 4 groups, and each group is 5. Wherein, 1-3 groups are safety test groups, each neck intramuscular injection vaccine is 4.0ml, the 4 th group is a negative control group, the neck intramuscular injection sterile physiological saline is 4.0ml, and the test is observed for 14 days.
1.3 efficacy test
Healthy and susceptible piglets of 14 days old (bordetella bronchiseptica agglutination detection antibody negative; pig D toxigenic Pasteurella multocida PMT blocking ELISA antibody is negative, and the PCR detection antigens of the nose swab and the pig B.multocida are negative) 20 heads, and the pigs are divided into 4 groups, and each group is 5 heads. Wherein 1-3 groups are immune groups, 1ml of vaccine is intramuscular injected into each head and neck of the piglet, and the piglet is subjected to secondary immune treatment in the same dosage and mode after 14 days; group 4 served as a control group without inoculation. All pigs were challenged 28 days after the second immunization, and 0.5ml of each nostril of each pig was slowly dropped into the solution containing 8.2X10 6 CFU/ml of B.bronchiseptica; toxin-producing pasteurella multocida attacks the toxin 28 days after the second immunity, the toxin attack method is the same as that, and each pig is slowly dripped into 0.5ml containing 1×10 of toxin 9 CFU/ml toxigenic Pasteurella multocida was used for 4 days, all experimental pigs were all killed for 14 days after challenge, the nose of the pigs was transected, the turbinate lesions were examined and scored, and the presence or absence of lesions was observed by section.
Porcine turbinate lesion scoring criteria: the test pigs were sacrificed, placed in a recumbent position, and their heads were slightly raised, and the kiss were cut laterally between the first and second molars, and the conditions of Turbinate Atrophy (TA) and Nasal Septum Deflection (NSD) were observed in the cross section. Each turbinate curl atrophy can be judged as 0-4 minutes (0 minutes: normal without any lesions; 1 minute: very small part of atrophy, one curl will shrink nearly half; 2 minutes: slight atrophy, one curl more than half atrophy; 3 minutes: moderate atrophy, turbinate rigidity; 4 minutes: severe atrophy, turbinate complete curl disappeared). The deflection of the nasal septum can be judged as 0 to 2 points (0 points: normal; 1 point: slight deflection; 2 points: severe deflection). The score of each pig turbinate lesion was 4 turbinate curl atrophy scores and the nasal septum deflection score were added, with a maximum score of 18 lesions.
2. Laboratory test results
2.1 physical Property inspection
Random sampling from 3 batches of vaccine was used for trait testing. The product is semitransparent suspension, has a small amount of sediment at the bottom after long-term placement, and is uniform suspension after shaking (see table 2).
Table 23 batch laboratory finished product property test results
2.2 sterility testing
5 bottles of 3 batches of vaccines are randomly and respectively taken, respectively inoculated with TG culture medium, cultured for 3 days at 37 ℃, respectively inoculated with thioglycolate culture medium and casein culture medium inclined planes, respectively cultured at 37 ℃ and 25 ℃, and subjected to aseptic growth (see Table 3) of all (5/5) after 7 days of culture, thereby meeting the requirements.
TABLE 3 results of vaccine sterility test for each batch
| Lot number | Sterility testing |
| 202001 | 5/5 aseptic growth |
| 202002 | 5/5 aseptic growth |
| 202003 | 5/5 aseptic growth |
2.3 inspection of the load
5 bottles were randomly and separately taken from 3 batches of vaccine, and the results were all satisfactory (Table 6). Wherein, 202001 batch detection 5 bottles of sampling detection products have the filling amount of 20.5 mL, 21.2 mL, 20.8 mL, 20.6 mL and 21.1mL respectively; 202002 the filling amount of 5-bottle sampling inspection products is 20.8, 21.5, 20.6, 20.5 and 20.9mL respectively; 202003 and 5-bottle sampling inspection products with the filling amounts of 20.4, 20.6, 20.9, 20.7 and 21.1mL (see Table 4) meet the requirements.
TABLE 4 results of vaccine load checks for each batch
| Lot number | Filling amount |
| 202001 | 20.2mL、20.3mL、20.5mL、20.6mL、21.1mL |
| 202002 | 20.6mL、21.2mL、20.6mL、20.2mL、20.6mL |
| 202003 | 20.3mL、20.6mL、20.7mL、20.5mL、21.2mL |
2.4 examination of residual Formaldehyde
And 5 bottles of 3 batches of vaccines are randomly and respectively taken, and formaldehyde residual quantity is detected. The formaldehyde residues of the 202001, 202002 and 202003 batches of vaccine were tested to be 0.11%, 0.10% and 0.13% (see table 5), respectively, as desired.
TABLE 5 determination of residual Formaldehyde amount of vaccine for each batch
| Lot number | Residual amount of formaldehyde |
| 202001 | 0.09％ |
| 202002 | 0.12％ |
| 202003 | 0.11％ |
2.5 Security check
The prepared three batches of vaccine are respectively injected into the neck muscle of each 14-day-old healthy susceptible piglet by 4.0ml, and no local and systemic adverse reaction caused by the vaccine occurs after 14 days of observation (see Table 6).
TABLE 6 safety test results of one single dose vaccination of 14 day old piglets
2.6 efficacy test
After the virus is removed, the pigs in the non-immune virus removing control group and the 3 vaccine immune groups have individual pigs with 1-2 days of body temperature rise, and the rest most pigs have no obvious abnormal clinical manifestation. After the immunization, the pigs of the immune group have no obvious difference in daily gain from a blank control group (P is more than 0.05) after the immunization, but are obviously higher than those of a non-immune immunization, the pigs of the immunized group have slight spiral atrophy on the turbinate part, the difference from the blank control group is not obvious, the turbinate atrophy of the challenge control group is serious, the lung lesions are obvious, and the difference from the blank control group is obvious (P is less than 0.05) (see table 7).
Table 7 3 statistical results of clinical symptoms after immunization of pigs with vaccine against virulence (I)
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Remarks: the superscript of different letters indicates that the difference is significant
Table 7 3 statistical results of clinical symptoms after immunization of pigs with vaccine and detoxification (II)
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The physical properties, sterility test, safety test, efficacy test and the like of 3 batches of porcine atrophic rhinitis inactivated vaccines are respectively tested according to tables 2 to 7, and the vaccine meets the requirements, has better protection capability on the attack actions of porcine bordetella bronchiseptica and toxigenic pasteurella multocida, and the vaccines prepared according to the examples 1, 2 and 3 meet the design.
Although the invention has been described hereinabove with reference to embodiments, various modifications thereof may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the features of the disclosed embodiments may be used in any combination with each other as long as there is no structural conflict, and the present disclosure is not to be taken in an exhaustive sense only for the sake of brevity and resource saving. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Sequence listing
<110> Jiangsu nan Dengya high technology Co., ltd
<120> an inactivated vaccine for atrophic rhinitis of pigs and preparation method thereof
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Claims (4)
1. An inactivated vaccine composition for atrophic rhinitis of pigs is characterized by comprising an antigen and an adjuvant, wherein the antigen consists of inactivated porcine toxigenic pasteurella multocida, inactivated porcine bordetella bronchiseptica, toxigenic pasteurella multocida PMT toxin N-terminal protein and toxigenic pasteurella multocida PMT toxin C-terminal protein;
the nucleotide sequence of the N-terminal protein of the toxigenic multi-killing Pasteurella PMT toxin is SEQ ID NO. 1, and the nucleotide sequence of the C-terminal protein of the toxigenic multi-killing Pasteurella PMT toxin is SEQ ID NO. 2;
the strain ZXT+Pm of the pig toxigenic pasteurella multocida has been preserved with the preservation number: cctccc No: m20211427;
the bordetella bronchiseptica AHBb strain has been deposited with the accession number: cctccc No: m20211379;
the adjuvant is carbomer water adjuvant.
2. The inactivated vaccine composition for atrophic rhinitis of pig according to claim 1, wherein the content of the toxigenic pasteurella multocida in pig is not less than 1 x 10 9 CFU/ml, the content of the porcine bordetella bronchiseptica is not lowAt 1X 10 9 CFU/ml, wherein the content of the N-terminal protein of the toxigenic pasteurella multocida PMT toxin is 10-80 mug/ml, the content of the C-terminal protein of the toxigenic pasteurella multocida PMT toxin is 10-80 mug/ml, and the ratio of the N-terminal protein of the toxigenic pasteurella multocida PMT toxin to the C-terminal protein of the toxigenic pasteurella multocida PMT toxin is 1:1.
3. A method for preparing an inactivated vaccine composition for swine atrophic rhinitis according to any one of claims 1 to 2, comprising the steps of:
s1: respectively and optimally culturing the swine toxigenic pasteurella multocida and the swine bronchogenic bordetella, and inactivating and removing endotoxin before the swine toxigenic pasteurella and the swine bronchogenic bordetella are cultured until the swine toxigenic pasteurella and the swine bronchogenic bordetella do not enter a platform stage; respectively cloning and expressing a toxigenic pasteurella multocida PMT toxin N-terminal protein and a pasteurella multocida PMT toxin C-terminal protein, and purifying;
s2: and mixing the swine toxigenic pasteurella multocida, the swine bronchogenic bordetella, the toxigenic pasteurella multocida PMT toxin N-terminal protein, the toxigenic pasteurella multocida PMT toxin C-terminal protein and the adjuvant to prepare the vaccine.
4. Use of an inactivated vaccine composition for swine atrophic rhinitis according to any one of claims 1 to 2 in the preparation of a medicament for preventing progressive or non-progressive swine atrophic rhinitis.
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| JP2004131417A (en) * | 2002-10-10 | 2004-04-30 | Nippon Inst For Biological Science | Vaccine preparation for swine atrophic rhinitis |
| CN102302771A (en) * | 2011-07-13 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Polyvalent inactivity vaccine for preventing and treating atrophic rhinitis of swine |
| CN105797152A (en) * | 2014-12-31 | 2016-07-27 | 普莱柯生物工程股份有限公司 | Vaccine composition and its preparation method and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2004131417A (en) * | 2002-10-10 | 2004-04-30 | Nippon Inst For Biological Science | Vaccine preparation for swine atrophic rhinitis |
| CN102302771A (en) * | 2011-07-13 | 2012-01-04 | 普莱柯生物工程股份有限公司 | Polyvalent inactivity vaccine for preventing and treating atrophic rhinitis of swine |
| CN105797152A (en) * | 2014-12-31 | 2016-07-27 | 普莱柯生物工程股份有限公司 | Vaccine composition and its preparation method and use |
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