HCV NS3/4A丝氨酸蛋白酶在大肠杆菌中表达、纯化

Expression and Purification of HCVNS3/4A Protease in the E.coli

在大肠杆菌中HCV NS3/4A丝氨酸蛋白酶获得表达并进行纯化。根据NS3与NS4A两者形成异源二聚体的结构要求,并参照文献,通过基因拼接的方法设计了单链NS3/4A丝氨酸蛋白酶的基因。合成相应引物,利用反转录PCR从HCV患者血液中扩增出该蛋白酶的编码基因,克隆入原核表达载体pRSET-A,将重组表达质粒pRSET-A-ns3/4a转化BL211(DE3)大肠杆菌,并诱导表达与纯化。成功地构建了重组表达质粒pRSET-A-ns3/4a;重组质粒转化的BL21工程菌经IPTG诱导表达后,表达产物经SDS-PAGE和Western-blot证实为NS3/4A丝氨酸蛋白酶,并用镍离子亲和层析方法获得纯化蛋白酶。获得的纯化NS3/4A丝氨酸蛋白酶为建立其酶活性测定系统奠定了基础。

In order to expression and purify HCVNS3/4A protease in the E. coll. methods, on the basis of NS3/ 4A protease required for heterogenous dimer, the single chain NS3 and NS4A gene fused by the linker was constructed. The HCV NS3/4A gene was amplified by RT-PCR from the HCV positive serum. This fragment digested by BamH I / Hindm was cloned into pRSET-A by T4 ligase and transformed into E. coli. BL21 （DE3） ,then the recombinant plasmid named pRSET-A-ns3/4a was constructed successfully. The results show that the recombinant NS3/4A protease was expressed in the transformants distinctly after inducing by IPTG . The product was identified as NS3/4A protease by SD-PAGE and western-blot analysis . The expression production was purified on Ni^2＋ -NTA column . The purified NS3/4A protease could further be used to develop the enzymatic assay.