摘要
目的:阐明丙型肝炎病毒(HCV)非结构蛋白NS5A(NS5A)反式激活基因NS5A-TP9表达的调控机制。方法:选取NS5A-TP9基因翻译起始密码子ATG上游661 bp的基因组DNA序列作为启动子序列,应用聚合酶链反应(PCR)技术,以肝母细胞瘤细胞系HepG2基因组DNA为模板,扩增该启动子DNA片段,将其克隆至pCAT3中,构建pCAT3-NS5A-TP9-promoter报告基因表达载体,以该质粒转染HepG2细胞,用酶联免疫吸附法(ELISA)检测报告基因编码产物氯霉素乙酰转移酶(CAT)的表达活性。结果:发现质粒pCAT3-NS5A-TP9-promoter具有指导报告基因CAT转录表达的启动子活性,CAT的吸光度(A值)是缺乏启动子序列对照质粒pCAT3的13.9倍。结论:本研究克隆的启动子DNA序列具有指导下游基因转录的启动子活性,这一结果为研究新基因NS5A-TP9转录表达的调节机制,进一步阐明丙型肝炎病毒非结构蛋白NS5A的作用机制奠定了基础。
Objective: To clarify the expression and regulation mechanism of the new target gene (NS5A-TP9) transacti-vated by nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) .Methods: Six hundred and sixty-one base pairs (bp) up-stream of the translation start site was selected as promoter sequence which was amplified from hepatoblastoma cell line-HepG 2 cell genomic DNA by polymerase chain reaction (PCR) .The amplified product was cloned into pCAT3 vector.The HepG2 cell line was tranfected by pCAT3-NS5ATP9-promoter.The CAT activity was detected by an enzyme-linked immunosorbent assay (ELISA) kit. Results: We found pCAT3-NS5ATP9-promoter had high activity of CAT expression.The expression of CAT was 13.9 times as higher as the negative control plasmid pCAT3-basic.Conclusion: These results indicated that the pCAT3-NS5ATP9-promoter has promoter activity.
出处
《中西医结合肝病杂志》
CAS
2003年第6期357-359,共3页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
基金
国家自然科学基金(No.C39970674
No.C03011402
No.C39900130
No.C30070689)
