含人过氧化物酶体增殖物激活受体δ基因高效真核表达载体的构建及其意义

THE CONSTRUCTION AND SIGNIFICANCE OF THE HIGHLY EFFICIENT EUKARYOTIC EXPRESSION VECTOR CARRYING HUMAN PPARδ GENE

目的构建高效表达人过氧化物酶体增殖物激活受体δ(hPPARδ)的真核表达载体,为hPPARδ受体功能和基于hPPARδ受体靶点的药物筛选提供分子研究平台。方法采用逆转录-聚合酶链式反应(RT-PCR),从HepG2细胞总RNA克隆hPPARδ全长基因,与经BamHI、SalI相同双酶切的pIRES2-EGFP载体连接,构建重组质粒phPPARδ-IRES2-EGFP,经酶切及基因测序鉴定重组质粒中hPPARδ基因的完整性和忠实性;荧光显微镜观察重组质粒转染的293细胞GFP报告基因表达强度,并对转染细胞hPPARδ的表达进行荧光定量PCR和免疫细胞化学检测。结果经酶切和测序证实重组质粒构建正确,并在转染的293细胞中获得hPPAR6的高效表达。结论成功构建phPPARδ-IRES2-EGFP重组质粒。

Objective To construct a highly efficient eukaryotic expression vector carrying human peroxisome proliferator- activated receptor δ（hPPARδ） gene in order to provide an ideal molecular platform for screening natural ligands and functional study of hPPARδ. Methods hPPARδ gene, cloned from total RNA of HepG2 cells by RT-PCR, was ligated with pIRES2-EGFP plasmid which was excised by BarnHI and Sa/I double endonucleases. The recombinant plasmid was transfected into 293 cells. Real-time quantitative PCR and immunocytochemistry assays were used to analyze the expression levels of hPPARδ in the transfected 293 cells. Results hPPARδ gene sequence contained in the recombinant plasmid phPPARδ-IRES2-EGFP was verified correct by enzyme digestion as well as sequence analysis. After being transfected into 293 cells, high efficient expression of hPPARδ gene contained in phPPARδ-IRES2-EGFP plasmid was found to display a highly efficient expression detected both at mRNA and protein levels by real-time quantitative PCR and immunocytochemistry respectively. Conclusion The recombinant plasmid phPPARδ-IRES2-EGFP has been constructed successfully with a highly efficient expression in culture 293 ceils, which provides a useful tool in analyzing hPPARδ gene＇s function as well as establishing a molecular platform by which the candidates of unknown hPPARδ ligands or activators can be found.