一种新的丙型肝炎病毒单链丝氨酸蛋白酶基因的构建、表达与鉴定

Construction, Expression and Identification of Gene of a Novel Single-chain Serine Protease of Hepatitis C Virus in E.coli

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 .

Based on the crystallographic structure of hepatitis C virus(HCV) serine protease, a novel single-chain serine protease was designed in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR carried out with three primers and then cloned into the prokaryotic expression vector pQE30. The recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was over-expressed as soluble protein when the \%E.coli\% was induced at low dosage of IPTG and cultured at low temperature. The protein was purified to homology using Ni-NTA agarose metal affinity resin. The purity was estimated to be over 95%. The purified protein was used as antigen to detect the specific antibodies in blood donors. It was found that over seventy-four percent of blood donors had specific antibodies. A cleavage system was established with recombinant substrate NS5ab and purified protease in vitro. When it was used as model to evaluate some compounds, PMSF was found to inhibit the activity of serine protease, while the EDTA was not. Thus, the purified single-chain serine protease and the \%in vitro\% cleavage system make it possible to develop enzyme inhibitors.