为获得猪脑心肌炎病毒(EMCV)3C蛋白并进一步了解其结构,本研究以EMCV HB10株的RNA为模板,通过RT-PCR扩增3C基因,将其克隆至pMD18-T载体,经PCR、双酶切和测序鉴定,挑选测序正确的质粒经双酶切获得目的片段并将其克隆至pET32a载体以构建重组原核表达质粒.将重组质粒转化至大肠杆菌TranSetta(DE3)感受态细胞,经IPTG诱导后,SDS-PAGE检测目的蛋白表达情况,Western blotting检测纯化后3C蛋白及其对EMCV的反应原性.结果显示,成功克隆3C基因,长度为615 bp,编码205个氨基酸.SDS-PAGE结果显示,3C蛋白主要以包涵体形式表达,His-3C蛋白大小约为40 ku.Western blotting分析可知,纯化后His-3C蛋白条带单一,同时3C蛋白与EMCV细胞毒有良好的反应原性.经生物分析软件预测可知,EMCV 3C蛋白为非分泌蛋白,无跨膜区,有多个磷酸化位点,参与蛋白的水解过程.3C蛋白酶作为EMCV基因组编码蛋白中唯一的蛋白酶,在病毒复制的过程中具有不可或缺的作用.本研究成功构建了EMCV 3C蛋白原核表达载体并对其结构进行了预测,为进一步研究3C蛋白的作用及催化机理奠定基础.%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.
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