含PGK启动子慢病毒表达质粒的构建与鉴定

Construction and Identification of the Lentiviral Expression Vector With Human PGK Promoters

为了构建一个含有人源PGK1(human phosphoglycerate kinase 1)启动子的慢病毒表达载体pL-PGK-GFP.采用PCR从人组织中扩增PGK1基因的启动子部分,再用酶切-连接的方法将扩增的启动子区片段亚克隆入慢病毒表达质粒pL-EGFP中,再用测序、酶切和瞬时表达的方法进行鉴定.结果是下游的eGFP基因在PGK1启动子驱动下,在293FT细胞中表达绿色荧光蛋白报告基因,这表明成功构建了慢病毒表达质粒pL-PGK-GFP.扩增的537bp PGK1启动子片段具有一定的启动效率,能在HIV来源的慢病毒载体中驱动下游目的基因的表达.

To construct a lentiviral vector pL-PGK-GFP containing the promoter of human phosphoglycerate kinase 1 （PGK1）, the promoter fragment of the PGK1 was amplified by PCR from human tissue, then the promoter fragment was subcloned into the lentiviral vector pL-GFP, and was confirmed by double restriction enzyme digestion, sequencing and transiently expressing. The results indicated that the green fluorescence was expressed in 293FT cells by the downstream eGFP gene that was driven by PGK1 promoter, which confirmed that the lentiviral vector pL-PGK-GFP was constructed successfully. The downstream target gene can be driven to express by amplified 537bp PGK1 promoter which promote efficiently in the lentiviral vector of the HIV- based.