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根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .
A novel single-chain serine protease molecule was designed and constructed based on the structural characteristics of hepatitis C virus (HCV) serine protease, which consists of the core sequence of cofactor NS4A, the flexible linker GSGS and the NS3 serine protease domain .According to the designed three primers, the gene of single-stranded serine protease was obtained by two rounds of PCR and inserted into the prokaryotic expression vector pQE30 and transformed into E.coli M15 to obtain the recombinant clone.After low-dose induction and low-temperature culture, the target gene was highly soluble The purity of the recombinant protein purified by metal chelation chromatography was over 95% .The indirect ELISA assay of 98 serum samples confirmed that the recombinant protein had good antigenicity and specificity. The recombinant protein substrate NS5ab and single-chain serine protease A simple and practical serine protease in vitro activity assay system was established, and the effect of PMSF and EDTA on protease activity was observed by this system.The results showed that PMSF could inhibit the enzymatic activity of protease, while EDTA could not inhibit the activity of the enzyme. The successful expression of HCV serine protease and establishment of an in vitro activity assay system for the serine egg Developed inhibitors laid the material foundation.