Establishment of stably transfected HepG2 cell line expressing HCV scNS4A/NS3 protease

    LEI Yingª²Feng, YIN Wen, XUE Xiaoª²Ping, YANG Jing, L¦a Xin, WEI Sanª²Hua, HU Xingª²Bin, SUN Mengª²Ning£¬ XU Zhiª²Kai

    Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi¬ðan 710033£¬China

    ¡¾Abstract¡¿ AIM: To construct a eukaryotic expression vector of HCV single chain serine protease (scNS4A/NS3) and to obtain its stably transfected HepG2 cell line. METHODS: According to the sequence of HCV scNS4A/NS3 gene from the literature, the primers amplifying the gene coding scNS4A/NS3 protease were designed. HCV RNA was extracted from the HCV positive serum and the gene coding scNS4A/NS3 protease was amplified via RTª²PCR. The PCR product was digested by BamH¢ñ/Hind¢ó and purified by gel extraction. This fragment was inserted into pcDNA3.1(-) with T4 ligase and transformed into E.coli JM109. The positive recombinant plasmid was selected and identified via sequence assay and restrictive enzyme digestion. The recombinant plasmid was then transfected into HepG2 cell by LipofectAMINE2000. The cells containing stable transformants were selected by the ability of resistance to G418. The stably transfected cell line was identified by RTª²PCR and IFA and Westernª²blot. RESULTS: The eukaryotic expression vector named pcDNA3.1(-)ª²scNS4A/NS3 was successfully constructed and the stably transfected HepG2 cell line which expressed scNS4A/NS3 protease was obtained. CONCLUSION: The stably transfected HepG2 cell line expressing single chain serine protease facilitates the establishment of cellª²based system in evaluating antiª²HCV serine protease drug. ......