重组人PPARα基因载体phPPARα-IRES2-EGFP高效体外转染表达体系的建立  

Establishment of high efficient transfection and expression system of phPPARα-IRES2-EGFP recombinant plasmid carrying human PPARα gene in vitro

作　　者：章涛[1,2] 万敬员[1] 刘颖菊[1] 杨俊卿[1] 周元国[3] 周岐新[1] 

机构地区：[1]重庆医科大学药理学教研室 [2]遵义医学院贵州省细胞工程重点实验室,贵州遵义563003 [3]第三军医大学创伤烧伤与复合伤国家重点实验室

出　　处：《中国现代医学杂志》2007年第17期2073-2077,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金资助项目(30572353)

摘　　要：目的建立体外phPPARα-IRES2-EGFP质粒高效转染并获得重组基因hPPARα高效表达的体系。方法采用阳离子脂质体Lipofectamine 2000将phPPARα-IRES2-EGFP转染入293细胞,荧光显微镜观察质粒转染的293细胞绿色荧光蛋白(GFP)报告基因表达强度及转染效率,并对转染细胞hPPARα的表达进行荧光定量PCR和Western Blot检测。结果荧光显微镜下可见转染phPPARα-IRES2-EGFP的293细胞GFP报告基因高表达,转染效率高达(83±9)%;并且,其hPPARαmRNA表达水平高于空载体转染对照组3个数量级,hPPARα蛋白表达也远高于对照组(P<0.01),说明导入的重组质粒能够高效表达hPPARα。结论成功建立重组质粒phPPARα-IRES2-EGFP高效体外转染表达体系,为hPPARα受体功能的研究及基于hPPARα为靶标的药物筛选平台的建立奠定了基础。[Objective] To establish high efficient transfection and expression system in vitro for phPPARα- IRES2-EGFP recombinant plasmid carrying human peroxisome proliferator-activated receptor （hPPARα） gene. [Methods] Cationic lipid transfection reagent lipofectamine 2000 was used to transfect phPPARα-IRES2-EGFP recombinant plasmid and control pIRES2-EGFP plasmid to 293 cells. The profiles of GFP expression and transfection efficiency were measured under fluorescence microscopy. Expression of PPARα in transfected 293 cells was determined by western blot and real time quantitative polymerase chain reaction（PCR）. [Results] GFP expression level was high in transfected 293 cells, and the transfection efficiency of phPPARα-IRES2-EGFP was （83±9）%. In transfected 293 cells, high efficient expression of hPPARα gene was detected both at mRNA and protein levels by realtime PCR and western blot analysis. [Conclution] Successful establishment of high efficient transfection arid expression system for phPPARα-IRES2-EGFP recombinant plasmid in vitro, which provide a useful tool in studying hPPA-Rα gene function as well as establishing molecular platform by which the unknown hPPARα activators can be found.

关 键 词：过氧化物酶体增殖物激活受体 转染 真核表达 293细胞 

分 类 号：R966[医药卫生—药理学]