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

发布时间：2018-05-16 00:43

本文选题：脑心肌炎病毒 + C蛋白酶　；参考：《中国畜牧兽医》2017年06期

【摘要】：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615bp,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40ku。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。
[Abstract]:In order to obtain the protein of porcine encephalomyocarditis virus (EMC V3 C) and further understand its structure, the RNA of EMCV HB10 strain was used as template, the 3C gene was amplified by RT-PCR, cloned into pMD18-T vector, and identified by PCR, double enzyme digestion and sequencing. The target fragment was obtained by double enzyme digestion and cloned into pET32a vector to construct the recombinant prokaryotic expression plasmid. The recombinant plasmid was transformed into E. coli tranSetta-DE3) competent cells. The expression of the target protein was detected by SDS-PAGE after IPTG induction. The purified 3C protein and its reactivity to EMCV were detected by Western blotting. The results showed that 3C gene was cloned successfully, the length was 615bpand encoded 205 amino acids. SDS-PAGE showed that the size of His-3C protein expressed mainly in inclusion body was about 40ku.Western blotting, and the His-3C protein band was single after purification. At the same time, 3C protein has good reactivity with EMCV cytotoxicity. According to the prediction of bioanalysis software, EMCV3C protein is a non-secretory protein with no transmembrane region, and there are many phosphorylation sites. The protease. 3C protease is the only protease in the EMCV genome coding protein. It plays an indispensable role in the process of virus replication. In this study, the prokaryotic expression vector of EMCV 3C protein was successfully constructed and its structure was predicted, which laid a foundation for further study on the function and catalytic mechanism of 3C protein.
【作者单位】：中国农业科学院北京畜牧兽医研究所;
【基金】：中国农业科学院创新工程项目(ASTIP-IAS15)
【分类号】：S852.651

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