Construction of the promoter indentifying plasmid with GFP reporter gene and identification of the promoter activity of HCV 5'- UTR / 重庆医科大学学报

ABSTRACT

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.