Porcine epidemic diarrhea-porcine clostridial enteritis bivalent subunit oral vaccine and preparation method thereof

A technology for porcine epidemic diarrhea and oral vaccine, which is applied in the field of oral vaccine and preparation of porcine epidemic diarrhea-Clostridium suis enteritis dual subunit, can solve the problems of poor immune effect and large stimulation stress of immunized animals, and achieves Less impurity proteins, high mucosal antibodies, and the effect of changing immune pathways

Pending Publication Date: 2020-03-17
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The first object of the present invention is to provide an oral vaccine of porcine epidemic diarrhea-clostridium suis enteritis dual subunit, to alleviate the need to inject two kinds of vaccines respectively in the prior art, the immune effect is poor and the immunized animal stimulates the stress greatly technical issues

Method used

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preparation example Construction

[0044] The present invention also provides the preparation method of the above-mentioned oral vaccine, wherein the immune antigen is emulsified and mixed with the vaccine adjuvant and the oral adjuvant in sequence to obtain the porcine epidemic diarrhea-clostridium enteritis dual subunit oral vaccine. The preparation method is simple and easy to operate, does not need special production equipment, and the production cost of the oral vaccine is relatively low.

[0045] In a preferred embodiment, the inactivation methods of inactivating porcine epidemic diarrhea virus S protein and inactivating porcine Clostridium perfringens beta toxin protein independently include: using an inactivating agent to mix and inactivate at 2°C-8°C Live for 22-26 hours, then mixed and hydrolyzed at 36-38°C for 1-3 hours, and then sterilized to obtain inactivated porcine epidemic diarrhea virus S protein or inactivated porcine Clostridium perfringens beta toxin protein.

[0046] The inactivator can be...

Embodiment 1

[0053] This embodiment provides a method for expressing inactivated porcine epidemic diarrhea virus S protein, comprising the steps of:

[0054](a), codon optimization and synthesis of porcine epidemic diarrhea virus S protein, the amino acid sequence of porcine epidemic diarrhea virus S protein is shown in SEQ ID NO.1, porcine epidemic diarrhea virus PEDV-S gene codon optimization and synthesis Entrust a synthesis company to synthesize.

[0055] (b) Construction and identification of the peD-NA3-S recombinant plasmid, inserting the PEDV-S protein gene into the eukaryotic expression vector peD-NA3.1(+), and obtaining the recombinant plasmid peD-NA3-S.

[0056] (c), cell transfection, according to cell density 2×10 5 cells / ml, inoculate a six-well plate, 2ml per well, place at 37°C, 5% CO 2 Incubate overnight in the cell culture incubator; start transfection when the confluence of cells reaches 80%-90%, change the medium to DMEM / F12 without antibiotics and serum before transf...

Embodiment 2

[0063] This embodiment provides a method for expressing inactivated piglet Clostridium perfringens beta toxin protein, comprising the following steps:

[0064] (a), codon optimization and synthesis of Clostridium perfringens porcine beta toxin protein, the amino acid sequence of Clostridium perfringens porcine beta toxin protein is shown in SEQ ID NO.2, Clostridium perfringens porcine beta toxin protein The codon optimization and synthesis of the toxin CNE-β gene was commissioned by a synthetic company.

[0065] (b) Construction and identification of the peD-NA3-β recombinant plasmid, inserting the CNE-β protein gene into the eukaryotic expression vector peD-NA3.1(+) to obtain the recombinant plasmid peD-NA3-β.

[0066] (c), cell transfection, according to cell density 2×10 5 cells / ml, inoculate a six-well plate, 2ml per well, place at 37°C, 5% CO 2 Incubate overnight in the cell culture incubator; start transfection when the confluence of cells reaches 80%-90%, change the m...

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Abstract

The invention belongs to the technical field of biology and provides a porcine epidemic diarrhea-porcine clostridial enteritis bivalent subunit oral vaccine and a preparation method thereof. The porcine epidemic diarrhea-porcine clostridial enteritis bivalent subunit oral vaccine comprises two antigens, a vaccine adjuvant and an oral adjuvant, wherein the two antigens are an inactivated porcine epidemic diarrhea virus S protein and an inactivated porcine clostridium perfringens beta toxin protein respectively. With the vaccine adopted, a pig can simultaneously obtain the porcine epidemic diarrhea virus antibody and the porcine clostridium perfringens beta toxin antibody, so that the pig can be prevented from being infected with epidemic diarrhea and clostridial enteritis. The vaccine is taken orally, so that an immunization route is changed; the immunological stress of the pig can be reduced; the side effect of the vaccine is slight; and therefore, the immune effect of the vaccine is effectively improved. The preparation method of the oral vaccine provided by the invention is simple and easy to operate and does not need special production equipment. The production cost of the oralvaccine is lower.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a porcine epidemic diarrhea-clostridium suis enteritis dual subunit oral vaccine and a preparation method thereof. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae and belongs to the genus Coronaviridae. Porcine epidemic diarrhea is an acute intestinal infectious disease of piglets and finishing pigs caused by a virus. The disease is very similar to infectious gastroenteritis. In my country, it mostly occurs from December to January-February of the next year, and it is also reported in summer. It can occur in pigs of any age, the younger the age, the more serious the symptoms and the higher the mortality rate. Pigs of all ages can be infected. Suckling pigs, shelf pigs or fattening pigs have a high incidence rate, especially suckling pigs suffer the most, and the incidence rate of sows varies greatly, about 15-90%. Sick pigs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/295A61K39/215A61K39/08A61K39/39A61P31/04A61P31/14
CPCA61K39/12A61K39/08A61K39/215A61K39/39A61P31/04A61P31/14A61K2039/70A61K2039/542A61K2039/552C12N2770/20034
Inventor 贺笋杜久斌潘毅平唐慧芬范斌张慧敏
Owner 天康生物制药有限公司
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