胆汁酸膜受体TGR5真核表达载体的构建及在细胞中的表达

试验旨在构建胆汁酸膜受体TGR5真核表达载体，转染293T细胞并在其中表达。用RT-PCR技术从胎盘组织中得到TGR5基因，克隆至真核表达载体pCMV-EGFP中。双酶切和测序鉴定正确后，将pCMV-EGFP-TGR5瞬时转染293T细胞，并采用实时荧光定量PCR和Western blotting技术检测TGR5的表达。结果显示，本试验构建了TGR5真核表达载体pCMV-EGFP-TGR5；转染293T细胞后，荧光显微镜观察到绿色荧光表达；实时荧光定量PCR检测TGR5表达量显著增加；Western blotting结果显示有目的蛋白表达。结果表明，真核表达载体pCMV-EGFP-TGR5构建成功，转染293T细胞后在基因、蛋白水平上均表达TGR5受体。

Construction of Bile Acid Receptor TGR5 Eukaryotic Expression Vector and its Expression in Cell

The study was aimed to construct a membrane-bound bile acid receptor TGR5 eukaryotic expression vector, which transfected into 293T cell, and expressed in 293T cell. Total RNA was extracted from placenta tissue, and RT-PCR was performed to obtain the TGR5 cDNA, which was inserted into the eukaryotic expression vector pCMV-EGFP. The novel constructed plasmid was confirmed by restriction enzyme digestion and DNA sequencing, then 293T cell was transfected with pCMV-EGFP-TGR5, Real-time PCR and Western blotting were amplified to determine the expression of TGR5. The result showed that the eukaryotic expression vector of pCMV-EGFP-TGR5 was constructed successfully. Green fluorescent was examined under fluorescent microscope, and the expression of TGR5 was increased significantly in 293T cells. The eukaryotic expression vector pCMV-EGFP-TGR5 was constructed successfully and TGR5 was expressed in mRNA and protein levels in 293T cell after transfection.