论文部分内容阅读
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 of 5 ’untranslated region (5’UTR) DNA sequence of Hepatitis C virus (HCV) genome in HepG2 cells to understand the mechanism of HCV replication regulation. Methods Plasmids 5’UTR-Luc (+) / (-) and 5’UTR DNA sequences driving the expression of luciferase gene driven by the forward and reverse 5’-UTR sequences of HCV genome were constructed respectively. UTR-EGFP (+) / (-) were transfected into HepG2 cells respectively. The expression of luciferase was detected by dual luciferase assay system. The m RNA level of luciferase gene was detected by reverse transcription polymerase chain reaction The expression level of green fluorescent protein gene was observed under a microscope and compared with the corresponding control to confirm the promoter activity of 5’UTR DNA sequence of HCV genome. Results 5’UTR-Luc (+) showed luciferase expression significantly but lower than that of pGL3 control (Luc / R 0.690 ± 0.086, Luc / RL 4.210 ± 0.340) , While luciferase expression was not obvious in 5 ’UTR-Luc (-) and pGL3 enhancer (Luc / RL were 0.095 ± 0.008 and 0.044 ± 0. 005 respectively); reverse transcriptase-polymerase chain reaction Consistent with this, the luciferase gene mRNA was detected by 5 ’UTR-Luc (+), while 5’ UTR-Luc (-) was undetectable. A strong green fluorescence was observed for 5’UTR-EGFP (+), while a non-fluorescent expression of 5’UTR-EGFP (-). Conclusion The 5’U TR DNA sequence of HCV genome has obvious promoter activity, which can activate the expression of downstream genes and play an important role in the replication of HCV genome.