丙型肝炎病毒5′非翻译区 DNA 序列在 HepG2细胞中的启动子活性

The promoter activity of the DNA sequence corresponding to HCV 5′UTR in HepG2

目的 研究丙型肝炎病毒(HCV)基因组5′非翻译区(5′UTR)DNA 序列在 HepG2细胞中 的启动子活性，以了解 HCV 的复制调控机制。方法 分别构建 HCV 基因组5′UTR DNA 正反向序列驱动 虫荧光素酶基因表达的质粒5′UTR-Luc(+)/(-)和5′UTR DNA 序列驱动绿色荧光蛋白基因表达的质粒5′ UTR-EGFP(+)/(-)，分别转染 HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，逆转录 聚合酶链反应检测虫荧光素酶基因 m R N A 水平，荧光显微镜观察绿色荧光蛋白基因的表达水平，并与相应 对照作比较，来证实 HCV 基因组5′UTR DNA 序列的启动子活性。结果 5′UTR-Luc(+)有明显的虫 荧光素酶表达，但比 pGL3 control 表达水平低(Luc/R为0．690±0．086，Luc/RL 为4．210±0．340)，而5 ′UTR-Luc(-)和 pGL3 enhancer 无明显虫荧光素酶表达(Luc/RL 分别为0．095±0．008和0．044±0． 005)；逆转录聚合酶链反应结果与之相符，5′UTR-Luc(+)检测到虫荧光素酶基因 mRNA，而5′UTR- Luc(-)则未检测到。5′UTR-EGFP(+)观察到较强绿色荧光，而5′UTR-EGFP(-)无荧光表达。 结论 HCV 基因组5′U TR DNA 序列具有明显的启动子活性，能启动下游基因的表达，在 HCV 基因组复 制过程中有重要作用。

Objective To study the promoter activity in HepG2 cells of the DNA sequence corresponding to the HCV 5＇ UTR. Methods Plasmids, 5＇ UTR-Luc（＋） and 5＇ UTR-Luc（-） carrying the forward and reverse DNA sequences corresponding to the HCV 5＇ UTR respectively were constructed, and subsequently transfected into HepaG2 cells. The luciferase activity and the mRNA of the luciferase gene were then detected. The 5＇ UTR sequence was cloned into a GFP vector to make 5＇ UTR-EGFP, and then the GFP expression was confirmed by fluorescence microscopy. Results 5＇ UTR-Luc（＋） had an obvious luciferase activity whereas 5＇ UTR-Luc（-） had nearly no luciferase activity. The former had a high level of luciferase mRNA while the latter could not be detected. An intense green fluorescence expression was observed in the ceils transfected with the plasmid of 5＇ UTR-EGFP. Conclusion The forward DNA sequence corresponding to HCV 5＇ -UTR had an obvious promoter activity in hepG2 cells. It may play an important role in the replication of HCV.