带GFP的启动子鉴定质粒的构建及在HCV启动子鉴定中的应用

目的:利用已有质粒构建带GFP报告基因的启动子鉴定质粒,并用此质粒鉴定丙型肝炎病毒5非翻译区(HCV5'-UTR)启动基因表达的活性.方法:用限制性内切酶将质粒pEGFP-N1上的EGFP基因片断切下,与用相同酶切去除荧光素酶基因片断的PGL3 enhancer质粒大片断连接,得到带GFP的启动子鉴定质粒pEGFP enhancer.HCV 5'-UTR片断用PCR方法获得,插入pEGFP enhancer的多克隆区,构建HCV 5'-UTR驱动GFP表达质粒.应用脂质体转染技术转染HepG2细胞并观察荧光.结果:成功构建带GFP报告基因的启动子鉴定质粒,并在此基础上构建了HCV 5'-UTR启动GFP表达的质粒.前者转染HepG2细胞后,几乎无GFP表达,而后者观察到较强的绿色荧光.结论:该实验构建的带GFP报告基因的启动子鉴定质粒本底表达低,可用于真核启动子的鉴定.HCV 5'-UTR可以启动报告基因的表达,具有启动子活性.

Construction of the promoter indentifying plasmid with GFP reporter gene and identification of the promoter activity of HCV 5'- UTR

Objective; To construct a promoter identifying plasmid with GFP as reporter gene, and then identify the promoter activity of HCV 5'- UTR with this construct. Methods: In order to construct the promoter identifying plasmid with GFP as reporter gene, the fragment of EGFP was obtained from the plasmid pEGFP - N1 by restrict enzymes digestion. And this fragment was combined with the larger fragment of plasmid PGL3 enhancer digested by the same enzymes. The fragment of HCV 5'- UTR was obtained by PCR amplification and inserted into the promoter identifying plasmid. The structure of constructed plasmid was confirmed by electrophoresis analysis and DNA sequencing. The function of construct was confirmed by lipofectamine - mediated transient expression in HepG2 cells. Results: DNA sequencing showed that the inserted fragment of the constructed plasmid was the same as the template HCV genome. The HepG2 cells transfected with promoter indentifying plasmid nearly didn' t express the reporter gene of GFP while the plasmid with HCV 5'- UTR could express the GFP gene. Conclusions; The results showed that the author successfully constructed the promoter identifying plasmid with GFP as reporter gene. The fragment of HCV 5'- UTR could behaviour as an eurakocyte promoter in HepG2 cells.