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Preparation method of porcine epidemic diarrhea virus genetic engineering subunit oral vaccine

A porcine epidemic diarrhea and genetic engineering technology, which is applied in the field of preparation of porcine epidemic diarrhea virus genetically engineered subunit oral vaccines, can solve the problems of bacterial virus harm to human body, inability to colonize intestinal mucosa, vaccine absorption, etc.

Active Publication Date: 2014-11-26
北京信得威特科技有限公司
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Problems solved by technology

[0006] However, the existing vaccines generally need to be injected. Routine injection of vaccines will cause stress and problems in vaccine absorption. In oral immunization, the immunogen may be degraded or inactivated before reaching the small intestinal mucosa. This purpose can be achieved as a living carrier, but bacterial viruses are harmful to the human body and cannot settle in the intestinal mucosa

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  • Preparation method of porcine epidemic diarrhea virus genetic engineering subunit oral vaccine
  • Preparation method of porcine epidemic diarrhea virus genetic engineering subunit oral vaccine
  • Preparation method of porcine epidemic diarrhea virus genetic engineering subunit oral vaccine

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preparation example Construction

[0024] see figure 1 , figure 2 with image 3 Shown, the preparation method of a kind of porcine epidemic diarrhea virus genetic engineering subunit oral vaccine of the present invention comprises the following steps:

[0025] 1).Synthetic M gene

[0026] Connect the synthetic M gene to the PES vector, carry out enzyme digestion according to the synthetic sequence instructions, clone them into pMD18-T plasmid vectors, transform competent JM109 cells, pick positive colonies, and extract a small amount of plasmids with the OMEGA kit for identification by enzyme digestion positive plasmid (see figure 1 );

[0027] 2). Construction of expression vector

[0028] The M gene fragment containing the correct direction and sequence was excised with restriction endonucleases EcoRI and SalI, recovered and purified from the gel, and then ligated with the vector plasmid pMG36e overnight at 16°C; the ligated products were mixed with competent cells MG1363 (laboratory Save) After mixing...

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Abstract

The invention discloses a preparation method of a porcine epidemic diarrhea virus genetic engineering subunit oral vaccine. The method comprises the following steps of: 1) synthesizing an M gene; 2) constructing an expression vector; 3) performing protein induction expression; 4) carrying out recombinant strain preservation experiment; and 5) preparing the vaccine. The oral immunization has the advantage of effectively stimulating local immune cells of the intestinal tract to generate secretion type IgA, is especially applicable to the infectious disease of the intestinal mucosa, and avoids stress and vaccine absorption problems resulting from conventional vaccine injection; and as a safe and non-toxic live vector system capable of living on the intestinal mucosa, the lactococcus lactis is the most suitable vector system. The oral vaccine performs antigen presentation through the gastrointestinal mucosa, and produces immune reaction similar to conventional injection through different immune pathways.

Description

technical field [0001] The invention relates to a preparation method of porcine epidemic diarrhea virus gene engineering subunit oral vaccine. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV) and is characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets. Intestinal infectious disease. PEDV and porcine transmissible gastroenteritis virus (TGEV) belong to the Coronavirus genus of the family Coronaviridae of the order Nidovirales, and the clinical symptoms caused by the two viruses are very similar , causing serious harm to the pig industry. [0003] PEDV directly enters the small intestine after being infected by the mouth and nose, and proliferates in the small intestinal epithelial cells, causing the degeneration, damage, shedding and atrophy of small intestinal villi absorbing epithelial cells, resulting in decreased...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/215
Inventor 李朝阳李明义石乔
Owner 北京信得威特科技有限公司
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