Protective antigen protein of porcine toxigenic pasteurella multocida as well as application and vaccine of protective antigen protein

A technology of protective antigen and Pasteurella, which is applied in the field of immunology, can solve the problems of low production capacity, unfavorable scale-up production, unsatisfactory protein expression, and increased production cost of the production line, so as to increase production practicability and good immunity Originality, conducive to the effect of production amplification

Pending Publication Date: 2022-03-25
河南兴华生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method needs to construct 4 kinds of recombinant strains, and carry out 4 lines of amplified production to express 4 kinds of proteins respectively, and then mix the 4 kinds of subunit proteins directly without purification and inactivate at 60°C to form a vaccine, and the endotoxin in the vaccine The content is high, which affects the safety of the vaccine. Too much foreign protein in the unpurified vaccine can easily cause stress in the pig herd, and the operation is cumbersome, which greatly increases the production cost of the production line. Moreover, the expression of the above four proteins is 212 -563μg / ml, the protein expression is not ideal, and the production capacity is low, which is not conducive to scale-up production

Method used

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  • Protective antigen protein of porcine toxigenic pasteurella multocida as well as application and vaccine of protective antigen protein
  • Protective antigen protein of porcine toxigenic pasteurella multocida as well as application and vaccine of protective antigen protein
  • Protective antigen protein of porcine toxigenic pasteurella multocida as well as application and vaccine of protective antigen protein

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Experimental program
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Effect test

Embodiment 1

[0032] Construction of embodiment 1 recombinant strain

[0033] 1.1 Construction of expression engineering bacteria

[0034] According to literature research: the full length of PMT is toxic, and PMT-N is more immunogenic than the C-terminal of PMT. Afterwards, we constructed 3 strains expressing the full-length of PMT, PMT-N (PMT N-terminal 1~ 520 fragment), PMT-C protein (PMT 520-1295 fragment), PET-PMT full-length (BL21), PET-PMT-N (BL21), PET-PMT-C (BL21) expression engineering bacteria .

[0035] 1.2 Full-length PMT, PMT-N, PMT-C protein expression, verification and purification quantification

[0036]Express engineering bacteria PET-PMT full-length (BL21), PET-PMT-N (BL21), PET-PMT-C (BL21) were streaked on LB (corresponding to resistance) solid plates and cultured at 37°C for 16 hours, and single The colonies were inoculated in 5ml LB liquid medium containing 100μg / ml corresponding resistance, cultured on a shaker (150r / min) at 37°C for 16h, and then inoculated at 1%...

Embodiment 2

[0038] Example 2. Vaccine Preparation

[0039] 2.1 Preparation of PMT full-length vaccine, PMT-N vaccine, PMT-C vaccine, PMT-N+PMT-C vaccine

[0040] Take a certain amount of PMT full-length protein (1), PMT-N protein (2), PMT-C protein (3) and PMT-N+PMT-C protein (4) prepared in Example 1 and add a certain amount of Bowers' protein Whole bacteria inactivated liquid, add a certain proportion of water-based adjuvant, stir evenly with a magnetic stirrer, and then sub-package. After passing the inspection, it is the porcine atrophic rhinitis subunit vaccine, wherein the antigen content in the vaccine is 50-200 μg / ml, The ratio of antigen and adjuvant in the vaccine is as follows:

[0041] Table 1 Composition of vaccines 1, 2, 3, 4

[0042]

[0043]

[0044] Note: "-" means no addition.

Embodiment 3

[0045] Embodiment 3. Vaccine sterility test

[0046] The sterility test was performed on the above three subunit vaccines in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia". The results were sterile growth.

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Abstract

The invention relates to the technical field of immunology, in particular to a protective antigen protein of porcine toxigenic pasteurella multocida as well as application and a vaccine of the protective antigen protein. The protective antigen protein is a polypeptide fragment from the first to the 520th of the N terminal of PMT protein. Experiments show that the protective antigen protein has high immunogenicity, and can effectively prevent and / or treat atrophic rhinitis of pigs. The invention further provides an escherichia coli expression system PET-PMT-N strain for expressing the antigen protein, 95% soluble protein can be obtained through expression and purification, the problems of inclusion body protein purification and renaturation are solved, the yield of target protein obtained through purification of PMT-N is increased to 0.4-0.8 mg / ml, the production capacity is greatly improved, and the vaccine is good in immune effect, high in safety and suitable for industrial production. The preparation process is simple, inactivation is not needed, operation is simple, and production amplification is facilitated.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular to a protective antigen protein of Pasteurella multocida and its application and vaccine. Background technique [0002] Porcine atrophic rhinitis (atrophic rhinitis, AR) is a chronic respiratory disease characterized by extensive expansion of nasal cavity and atrophy of mucous membrane, submucosa and bone tissue. , slow growth, increased feed-to-meat ratio, higher input costs, increased pig breeding culling rate, severe secondary infection can cause mycoplasma, meningitis or porcine reproductive-respiratory syndrome and other diseases, bringing pig breeding industry greater economic loss. [0003] According to the pathogen and pathogenic characteristics, the disease can be divided into non-progressive atrophic rhinitis (NPAR) and progressive atrophic rhinitis (PAR). Non-progressive atrophic rhinitis is mainly caused by Bordetella bronchiseptica, Bb is a Gram-negative bacterium,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/285C12N15/31C12N1/21C12N15/70A61K39/102A61K39/39A61P11/02A61P31/04C12R1/19
CPCC07K14/285C12N15/70A61K39/102A61K39/39A61P31/04A61P11/02
Inventor 牛旻杨瑞华李国强鲁璐肖红照姚菓贾宾程帅
Owner 河南兴华生物技术有限公司
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