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

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

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HCVNS5A基因 ,构建HCVNS5A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HCVNS5A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HCVNS5A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SV4 0病毒早期启动子。本研究为进一步克隆HCVNS5A蛋白反式激活的靶基因 。

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.