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A method for preparing recombinant Pichia pastoris with pedv core antigen coe protein displayed on the surface

A technology of Pichia pastoris and core antigen, which is applied in the field of animal biomedical engineering, can solve the problems of inability to produce maternal immunity and the inability of circulating antibody IgG to provide protection, and achieve the effect that is beneficial to protein activity and maintenance

Active Publication Date: 2016-08-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A method for preparing recombinant Pichia pastoris with pedv core antigen coe protein displayed on the surface
  • A method for preparing recombinant Pichia pastoris with pedv core antigen coe protein displayed on the surface
  • A method for preparing recombinant Pichia pastoris with pedv core antigen coe protein displayed on the surface

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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. The design process of the upstream and downstream primers of the α-lectin gene AGα and the porcine epidemic diarrhea virus COE gene: designed with reference to the sequence of the α-lectin gene cDNA gene and the PEDV COE gene registered in GenBank, and the upstream primers were designed in the At the beginning of the gene, according to the restriction site on the expression vector pPICZ α A, a Kpn I restriction site was added to the upstream primer design of the AGα gene and 3 protective bases CGG were added before the Kpn I restriction site ; The downstream primer is designed at the end of the gene, and the Xba I restriction site is added, and 3 protective bases TGC are added before the Xba I restriction site; the upstream primer design of the COE gene adds EcoR I restriction site and three protective bases CCG are added before the EcoR I restriction site; the downst...

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 600nmAfter 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 / LNaHCO 3 , 5% hydrolyzed casein, 0.5% glucose sus...

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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 Patents(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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