猪脑心肌炎病毒非结构蛋白3C的原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2017.06.003

Abstract

Abstract:

To obtain the 3C protein of encephalomypcarditis virus (EMCV) and predict its structure,3C gene was amplified by RT-PCR with the RNA as template from HB10 strain. And the target gene inserted into pMD18-T vector. The plasmids were sequenced after verification by PCR and double enzyme digestion. The target gene was cleaved from the correct plasmid,and inserted into the pET32a vector. The recombinant pET32a-EMCV-3C was transformed into TranSetta (DE3) and then induced by IPTG. Then the size was identified by SDS-PAGE and the antigenicity was analyzed by Western blotting. According to the sequence results,the length of 3C gene was 615 bp,which encoded 205 amino acids. The results of SDS-PAGE showed that His-3C protein mainly existed in the form of inclusion body with the molecular of 40 ku. The Western blotting results verified that purified His-3C could react with rabbit anti-EMCV serum prepared with EMCV. Bioinformatics analysis indicated that 3C protein was non-secretory,and had multiple phosphorylation sites,but it had no transmembrane region. Thus,the 3C protein mainly involved in protein hydrolysis process.3C protease,as the only protease in the EMCV genome,played an indispensable role in viral replication. In this study,we successfully constructed the prokaryotic expression vector of 3C protein and predicted its structure,which provided a basis for further study of the role of 3C protein and catalytic mechanism.

Key words: encephalomypcarditis virus; 3C protease; prokaryotic expression; bioinformatics analysis

CLC Number:

S852.65

LUO Ya-kun, LIANG Lin, ZHU Yu, WANG Jing, LIU Qi, LIU Cun, CUI Shang-jin. Prokaryotic Expression and Bioinformatics Analysis of Nonstructure Protein 3C from Porcine Encephalomyocarditis Virus[J]. , 2017, 44(6): 1588-1595.

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Prokaryotic Expression and Bioinformatics Analysis of Nonstructure Protein 3C from Porcine Encephalomyocarditis Virus

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