猪流行性腹泻病毒COE基因在毕赤酵母中的表达及生物活性分析

doi:10.11843/j.issn.0366-6964.2014.08.020

Abstract

Abstract:

The aim of this study was to express the COE protein of porcine epidemic diarrhea virus (PEDV) in Pichia pastoris，and evaluate the neutralizing ability of mices serum after immunization with COE protein.A pair of primers was designed to clone PEDV COE gene，and the PEDV strain was isolated from a clinical sample.The PEDV COE ORF was amplified by RT-PCR.Then the PEDV COE gene was inserted into pPIC9K，and acquared the recombinant plasmid，pPIC9K-COE.After the pPIC9K-COE was linearized by SacⅠand transformed into P.pastoris GS115 by electroporation，the high copy positive recombinant bacteria were screened for inducible expression，which was induced by addition of methanol.SDS-PAGE and Western blot were conducted to analyze the recombinant protein and its immunogenicity.The neutralizing ability of mice serum after immuned with the recombinant PEDV COE was analyzed by virus neutralization test.An 530 bp gene, PEDV COE was amplified by RT-PCR，and the PEDV COE protein were expressed in P.pastoris.The result of SDS-PAGE showed that the 27 kDa PEDV COE protein was expressed in P.pastoris GS115，and the concentration was 30 mg•L^－1.Western blot analysis of the PEDV COE protein proved that the immunogenicity of the recombinant protein.And the virus neutralization test result showed that the sera from mice immunized with the recombinant protein possess neutralizing activity，and the neutralization titer was 1:36，while the control was less than 1:2.The results presented here demonstrate that PEDV COE protein can be correctly expressed in P.pastoris，and it has better biological activity.

CLC Number:

S852.659.6

HU Qing-song，LI Lyu-mu，ZHANG Xiao-fei，XU Fa-zhi，DING Xiao-ling，XU Yan-wei，DING Wei-min. Expression of Porcine Epidemic Diarrhea Virus COE Gene in Pichia pastoris and Bioactivity Analysis of the Recombinant Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(8): 1342-1347.

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Expression of Porcine Epidemic Diarrhea Virus COE Gene in Pichia pastoris and Bioactivity Analysis of the Recombinant Protein

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