丙型肝炎病毒核心蛋白6号结合蛋白基因启动子序列的确定及转录活性鉴定

Identification and evaluation promoter sequence and the transcription activation of Hcbp6 interaction with core protein of hepatitis C virus

目的研究丙型肝炎病毒(HCV)核心蛋白6号结合蛋白基因(Hcbp6)序列表达的调控机制。方法根据软件对启动子的预测,选取翻译起始密码子ATG上游3256bp及下游180bp的DNA序列, 分成5段活性区域,分别以聚合酶链反应技术(PCR),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-Hcbp6-p报告基因表达载体,将该质粒分别转染HepG2、NIH3T3细胞,用酶联免疫吸附法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-Hcbp6-1066p和pCAT3-Hcbp6-240p能够指导CAT的表达,其平均吸光度值(A)是pCAT3-basic对照质粒的3.1倍和6.4倍。结论本研究克隆的启动子DNA序列具有转录活性, 这一结果为研究Hcbp6的调节机制,进一步阐明HCV核心蛋白的作用机制奠定了基础。

Objective To clarify the expression and regulation mechanism of the new target gene （human hepatitis C virus binding protein 6, Hcbp6） interaction with the core protein of hepatitis C virus （HCV）. Methods Referring to the prediction online of the promoter region, 3 256 base pairs （bp） upstream and downstream of the translation start site were selected as 5 putative promoter sequences which were amplified from the hepatoblastoma cell line-HepG2 cell genomic DNA by polymerase chain reaction （PCR）. The amplified products were cloned into a pCAT3 vector. The HepG2 and NIH3T3 cell lines were transfected by pCAT3-Hcbp6-promoters. The CAT activity was detected using an enzymelinked immunoassay （ELISA） kit. Results We found that 2 kinds of pCAT3-Hcbp6-promoters （1066 and 240） could direct the reporter gene expression. The expression of CAT was 3.1 times and 6.4 times higher than that of the pCAT3-basic control vector. Conclusion These results indicated that pCAT3-Hcbp6-promoters have promoter activity.