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目的:构建丙型肝炎病毒单链丝氨酸蛋白酶(sc-NS4A/NS3)的真核表达载体;建立重组质粒稳定转染的HepG2细胞克隆.方法:根据文献报道设计扩增scNS4A/NS3编码基因的引物,从HCV阳性患者血清中提取病毒RNA,RT-PCR方法扩增出scNS4A/NS3基因片段,BamHⅠ/HindⅢ双酶切后连接到经同样酶切的真核表达载体pcDNA3.1(-),转化菌株JM109感受态细胞,获得重组质粒pcDNA3.1(-)~scNS4A/NS3,经酶切鉴定及序列测定.将阳性重组质粒用脂质体法转染HepG2细胞,经持续G418压力选择和克隆化获得稳定转染的细胞系,用RT-PCR,IFA,Western-blot证实该稳定细胞系可以表达单链丝氨酸蛋白.结果:成功构建了真核表达载体pcDNA3.1(-)-scNS4A/NS3;建立了稳定转染的HepG2细胞克隆,命名为scpHepG2.结论:获得稳定的scpHepG2细胞克隆可表达单链丝氨酸蛋白,为下一步建立以细胞为基础的评价抗HCV丝氨酸蛋白酶药物系统奠定基础.
Objective: To construct a eukaryotic expression vector for hepatitis C virus single-chain serine protease (sc-NS4A / NS3) and to establish a HepG2 cell clone stably transfected with the recombinant plasmid.Methods: According to the primers reported in the literature to amplify scNS4A / NS3 gene The viral RNA was extracted from the serum of HCV-positive patients. The scNS4A / NS3 gene fragment was amplified by RT-PCR and ligated into the eukaryotic expression vector pcDNA3.1 (-) digested with BamHⅠ / Hind Ⅲ. The recombinant plasmid pcDNA3.1 (-) ~ scNS4A / NS3 was obtained by restriction endonuclease digestion and sequencing.The positive recombinant plasmids were transfected into HepG2 cells by lipofectamine 2000. After continuous G418 pressure selection and cloning The stably transfected cell lines were obtained and confirmed by RT-PCR, IFA and Western-blotting.Results: The eukaryotic expression vector pcDNA3.1 (-) - scNS4A / NS3 was constructed successfully. The stable transfected HepG2 cell clone was constructed and named as scpHepG2.Conclusion: The stable scpHepG2 cell clone can express single-chain serine protein, which will lay the foundation for the next step to establish a cell-based drug system for anti-HCV serine protease.