猪流行性腹泻病毒CH/GX/2015/750A株S2蛋白的原核表达及抗原性分析

doi:10.16431/j.cnki.1671-7236.2020.06.004

Abstract

Abstract: The purpose of this study was to understand the antigenicity of porcine epidemic diarrhea virus (PEDV) S2 protein and lay a foundation for the further study of diagnostic kit and subunit vaccine.In this study,the partial fragment of S2 gene (S2A) of PEDV CH/GX/2015/750A strain was amplified by RT-PCR and inserted into prokaryotic expression vector pET-32a(+) to construct prokaryotic expression plasmid pET32a-S2A.The prokaryotic expression plasmid pET32a-S2A was transformed into E.coli BL21(DE3).The recombinant protein S2A was induced by IPTG and purified by affinity chromatography on Ni column.The activity of S2A protein was detected by Western blotting.The purified recombinant protein S2A was used to immunize Kunming mice to prepare polyclonal antibody.The titer of the polyclonal antibody was detected by indirect ELISA,and the specificity of the polyclonal antibody was verified by indirect immunofluorescence.The results showed that the optimal induction condition of recombinant S2A protein was that when the final concentration of IPTG was 0.2 mmol/L,the induction temperature was 37 ℃,and the induction time was 3 h.The recombinant protein mainly existed in the form of inclusion body.Western blotting results showed that the purified and renatured recombinant protein S2A could specifically bind to PEDV positive serum and had good react ogenicity.The titer of the polyclonal antibody was 1:32 000.IFA results showed that the polyclonal antibody could specifically recognize and bind PEDV virus.The results of this study showed that PEDV S2A protein had good antigenicity and could be used as a candidate antigen of diagnostic kit or subunit vaccine.

Key words: porcine epidemic diarrhea virus(PEDV); S2 protein; prokaryotic expression; antigenicity analysis; polyclonal antibody preparation

CLC Number:

S852.6

LIU Lei, QIN Yibin, LU Bingxia, JIANG Dongfu, CHEN Zhongwei, HE Ying, ZHAO Shuo, ZHOU Yingning, LI Bin, DUAN Qunpeng, LIANG Jiaxing, ZHAO Wu. Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(6): 1668-1676.

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Prokaryotic Expression and Antigenicity Analysis of S2 Protein of Porcine Epidemic Diarrhea Virus CH/GX/2015/750A Strain

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