丙型肝炎病毒解旋酶在大肠杆菌中的表达纯化及鉴定

Expression Purification and Identification of HCV Helicase Gene in the E.coli

解旋酶(helicase)在HCV基因组复制中负责RNA的解链,在复制中起着关键的作用。利用反转录PCR,从HCV患者血液中扩增出解旋酶基因,克隆入原核表达载体pProEXHTb,成功构建了HCV解旋酶原核表达质粒pProEXHTb-helicase。重组质粒转化DH5α大肠杆菌,经IPTG诱导表达后,表达产物经SDS-PAGE和Westernblot证实系解旋酶,并用镍离子亲和层析方法获得纯化解旋酶。

HCV helicase is responsible for unwinding of RNA genome and play a key role in the replication of HCV genome. The HCV helicase gene was amplified by RT-PCR from the HCV positive serum. This fragment digested by BamH Ⅰ / Hind Ⅲ was inserted into pProEXHTh by T4 ligase and transformed into E. coli. DH5α, then the recombinant plasmid named pProEXHTh-helicase was constructed successfully. Helicase was expressed distinctly after inducing by IPTG. The product was identified as Helicase by SD-PAGE and Western blot analysis. The expression production was purified on Ni^2＋ -NTA column.