Preparation method of recombinant pichia pastoris for surface display of PEDV core antigen COE protein

A Pichia pastoris, core antigen technology, applied in the field of animal biomedical engineering, can solve the problems that circulating antibody IgG cannot provide protection, cannot produce maternal immunity, etc., and achieve the effect that is beneficial to protein activity and maintenance

Active Publication Date: 2014-07-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Judging from the current research results and application effects on PEDV, TGEV and RV at home and abroad, piglets mainly obtain maternal sIgA through colostrum to produce passive immune protection against coronavirus diarrhea, and circulating antibody IgG in serum cannot provide protection. Parenteral immunity does not produce maternal immunity

Method used

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  • Preparation method of recombinant pichia pastoris for surface display of PEDV core antigen COE protein
  • Preparation method of recombinant pichia pastoris for surface display of PEDV core antigen COE protein
  • Preparation method of recombinant pichia pastoris for surface display of PEDV core antigen COE protein

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Embodiment 1

[0026] S1. Construction of recombinant plasmid pPICZ α A-COE-AGα for surface display of α lectin

[0027] S11. Design process of upstream primers and downstream primers of α-lectin gene AGα and porcine epidemic diarrhea virus COE gene: designed with reference to the sequences of the α-lectin gene cDNA gene and PEDV COE gene registered in GenBank, and the upstream primers were designed in the At the beginning of the gene, at the same time, according to the restriction site on the expression vector pPICZ α A, the upstream primer of the AGα gene was designed with Kpn I restriction site and in Kpn There are 3 protective bases CGG before the I restriction site; the downstream primer is designed at the end of the gene, and Xba I restriction site, in Xba There are 3 protective bases TGC before the I restriction site; the upstream primer of COE gene was added EcoR I restriction site and in EcoR There are 3 protective bases CCG before the I restriction site; the downstr...

Embodiment 2

[0048]A preparation method for preventing porcine epidemic diarrhea oral vaccine, comprising the steps of:

[0049] S1. Pick the identified positive transformants and inoculate them in 3 mL of YPD liquid, culture overnight at 30°C with shaking at 250 r / min; then inoculate them in 50 mL of BMGY (1% yeast extract, 2% peptone, 1mol / L Potassium phosphate buffer (pH6.0), 1.34% YNB (no yeast nitrogen source), 4×10 -5 % biotin, 1% glycerol) body culture medium, shaking culture at 30°C for 24 h, until the cell OD 600nm After reaching 2-6 hours, centrifuge to discard the supernatant, add 25 mL of BMMY liquid medium (1% yeast extract, 2% peptone, 1mol / L potassium phosphate buffer (pH6.0), 1.34% YNB (no yeast nitrogen source), 4 x 10 -5 % biotin, 0.5% methanol), continuous induction culture for 72 h.

[0050] S2. Cultivate to 72h to collect samples, centrifuge, remove supernatant, and recombined yeast with suspension buffer (0.2 mol / L NaHCO 3 , 5% hydrolyzed casein, 0.5% glucose su...

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Abstract

The invention relates to the field of the animal biomedical engineering and specifically discloses a preparation method of recombinant pichia pastoris for surface display of porcine epidemic diarrhea virus core antigen COE protein. A yeast alpha-agglutinin surface display system is adopted to express the antigen core gene, namely COE gene, of the PEDV, and the COE protein is anchored on the cell walls of the yeast, and therefore, the preparation method of recombinant pichia pastoris for surface display of porcine epidemic diarrhea virus core antigen COE protein is provided. The recombinant pichia pastoris can be prepared into novel vaccine products having excellent industrialization prospect; the popularization and application of the vaccines play a positive role in preventing viral diarrhea having serious harm to the pig industry and have great practical significance for protecting and promoting healthy development of the pig industry, and meanwhile, remarkable social and economic benefits can be achieved, and therefore, the vaccines have vast market and industrialization prospect.

Description

technical field [0001] The invention relates to the field of animal biomedical engineering, in particular to a method for preparing a recombinant Pichia pastoris that displays porcine epidemic diarrhea virus core antigen COE protein on its surface. Background technique [0002] Since October 2010, the incidence and prevalence of piglet diarrhea in major domestic pig-raising provinces have shown a continuous upward trend, and it is also an important cause of death of piglets less than 7 days old. The death toll of piglets in 10 provinces and cities exceeded 1 million, the incidence rate was 60%-100%, and the mortality rate was as high as 80%-100%, which seriously threatened the healthy development of the pig industry and caused huge economic losses. Porcine epidemic diarrhea virus (PEDV) is one of the main pathogenic factors recognized by scholars at home and abroad. The most effective way to prevent porcine viral diarrhea at present is to vaccinate. Although domestic vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/50C12N1/19A61K39/215A61P31/14C12R1/84
Inventor 马静云袁尧胡文锋李智丽曾喜多孙宝丽毕英佐
Owner SOUTH CHINA AGRI UNIV
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