绿色荧光蛋白和HCV5′NCR调控荧光素酶双顺反子真核表达载体的构建及其在肝细胞中的表达  

Construction of an expression vector with a bicistron of green fluorescent protein and HCV 5′NCR controlled luciferase gene and its expression in hepatocytes

作　　者：刘水平[1] 谭德明[1] 侯周华[1] 刘双虎[1] 

机构地区：[1]中南大学湘雅医院传染病研究所

出　　处：《湖南医科大学学报》2003年第1期20-22,共3页Bulletin of Hunan Medical University

基　　金：湖南省科技攻关项目 (0 2ssy3 0 83 ) ;湖南省医药卫生科技项目 (2 0 0 0 0 0 1)

摘　　要：目的 :探讨绿色荧光蛋白 (greenfluorescentprotein ,GFP)和HCV 5′NCR(noncodingregion)调控荧光素酶双顺反子真核表达载体的构建及其在肝细胞中的表达。方法 :用基因重组技术 ,将真核表达载体pCMVNCRLuc的HCV5′NCR及其调控的荧光素酶基因亚克隆至报告载体pEGFP C1的GFP基因下游 ,构建含GFP和HCV 5′NCR调控荧光素酶双顺反子的真核表达载体pGFPNCRLuc。用脂质体基因转染技术 ,将pGFPNCRLuc转染至肝细胞株QSG770 1。结果 :GFP和荧光素酶基因均得到高效表达 ,其荧光素酶表达强度与pCMVNCRLuc比较 ,差异无显著性 (P >0 .0 5 ) ,GFP表达水平与pEGFP C1比较 ,差异无显著性 (P >0 .0 5 )。结论 :成功构建HCV和HCV 5′NCR调控荧光素酶双顺反子真核表达载体 ,HCV和荧光素酶基因在肝细胞内得到共表达。Objective To construct an expression vector with a bicistron of green fluorescent protein (GFP) and HCV 5′NCR controlled luciferase gene and to explore its expression in hepatocytes. Methods With molecular cloning techniques, the DNA fragment of HCV 5′NCR and its controlled luciferase gene was obtained from plasmid pCMVNCRLuc and subcloned into pEGFP C1, an expression plasmid of GFP. A recombinant plasmid pGFPNCRLuc with a bicistron of GFP and HCV 5′NCR controlled luciferase gene was constructed, and liposome mediated transient expression was detected in hepatocyte QSG7701 cells. Results The recombinant plasmid expressed both GFP and luciferase at a high level. The expression activity of GFP showed no significant difference compared to that of pEGFP C1 (P>0.05), nor did that of luciferase to pCMVNCRLuc (P>0.05). Conclusion An expression vector with a bicistron of GFP and HCV 5′NCR controlled luciferase gene was successfully constructed. GFP and luciferase could express independently. This study can promote the establishment of an anti HCV drug screen system.

关 键 词：丙型肝炎 绿色荧光蛋白 荧光素酶 QSG-7701细胞 

分 类 号：R346[医药卫生—基础医学]