丙型肝炎病毒非结构蛋白NS5A反式激活SV40病毒早期启动子的研究

目的：探讨丙型肝炎病毒(HCV)非结构蛋白NS5A的反式激活作用。方法：扩增HCV NS5A基因，构建HCV NS5A基因真核表达载体pcDNA3．1(-)—NS5A；并转染肝母细胞瘤细胞系HepG2细胞，免疫印迹方法检测转染细胞中HCV NS5A蛋白的瞬时表达；与报告质粒pCAT3--promoter共转染HepG2细胞，用酶联免疫吸附方法检测细胞中氯霉素乙酰转移酶(CAT)的表达活性。结果：质粒pcDNA3．1(-)—NS5A在HepG2细胞瞬时表达HCV NS5A蛋白，共转染实验中pcDNA3．1(-)—NS5A组的CAT表达活性是空质粒对照组的3．8倍。结论：构建的表达载体能在哺乳动物细胞中表达出相应蛋白，并能够反式激活SV40病毒早期启动子。本研究为进一步克隆HCV NS5A蛋白反式激活的靶基因，深入阐明HCV NS5A蛋白致肝细胞癌发生的分子生物学机制提供依据。

The study of transactivating effect of HCV NS5A protein on SV40 early promoter

Objective:To investigate the transactivating effect of hepatitis C virus (HCV) nonstructural protein 5A (NS5A).Methods: HCV NS5A gene was amplified from plasmid pBRTM3011 and the amplified product was cloned into pcDNA3.1( ) vector. Then the hepatoblastoma cell line HepG2 was transfected by pcDNA3.1( ) NS5A, and pcDNA3.1( ) NS5A and reporter plasmid pCAT3 promoter, respectively.HCV NS5A protein expressed in HepG2 cells was detected by Western blotting method. The activity of CAT was detected by a ELISA kit, which reflect the transactivating function of HCV NS5A protein. Results:HepG2 cells transfected with pcDNA3.1( ) NS5A can express HCV NS5A protein. The expression of CAT in HepG2 cells transfected with the pcDNA3.1( ) NS5A was 3.8 times as higher as that of control plasmid. Conclusion:It is suggested that the recombinant plasmid pcDNA3.1( ) NS5A can be expressed in mammalian cell line, and has transactivating effect on SV40 early promoter.