GGT1基因的真核表达载体构建及其表达定位

Construction of Eukaryotic Expression Vector of GGT1 and Observation of Its Subcellular Localization

构建GGT1重组真核表达载体,观察GGT1在COS7细胞中的定位。利用PCR技术扩增GGT1全长基因,经HindⅢ和EcoRⅠ酶切后连接入pEGFP-N1真核表达载体。将构建完成的重组表达质粒转染入COS7细胞,利用激光共聚焦显微镜观察GGT1在细胞中的定位。构建载体经双酶切和序列测定证实重组载体包含有正确的GGT1编码序列。激光共聚焦显微镜观察到,整个细胞内弥散绿色荧光,绿色荧光分布于COS7细胞浆中,提示GGT1定位于细胞浆中。成功构建人pEGFP-N1-GGT1真核表达载体,检测到GGT1定位于哺乳细胞的细胞浆中,为GGT1的功能研究提供了线索。

To construct the eukaryotic expression vector of GGT1 and detect its localization in COS7 cells.the full length GGT1 gene was amplified by PCR,and cloned into green fluorescence protein vector pEGFP-N1 to construct recombinant expression vector pEGFP-N1-GGT1.When recombinant plasmids were transfected into COS7 cells by lipofectimane 2000,the expression of GGT1 gene and its subcellular localization were observed with laser confocal microscope.Double restriction enzyme detection and DNA sequencing dectection suggested that the recombinant expression plasmid was correct.With the laser confocal microscope,the GGT1 fusion protein locates in cytoplasm of COS7 cel1s is observed.The recombinant expression vector pEGFP-N1-GGT1 is constructed successfully.GGT1 protein locates in cytoplasm of COS7 cells,which provides clues for the further function study of GGT1.