pGenesil-1-EGFP-shRNA/survivin真核表达载体构建及其表达验证

Construction and Expression Verification of Eukaryotic Expression Vector pGenesil-1-EGFP-shRNA/survivin

目的构建用survivin启动子驱动增强绿色荧光蛋白(EGFP)的shRNA真核表达载体pGenesil-1-EGFP-shRNA/survivin并验证其在宫颈癌Caski细胞中的表达。方法用有活性的survivin启动子取代pGenesil-1-EGFP-shRNA/U6载体中的U6启动子从而获得新的shRNA真核表达载体pGenesil-1-EGFP-shRNA/survivin;将pGenesil-1-EGFP-shBNA/survivin转染到宫颈癌Caski细胞及正常人脐静脉内皮HuVEC细胞,观察EGFP在两种细胞中的表达变化。结果成功构建pGenesil-1-EGFP-shRNA/survivin载体,分别转染Caski及HuVEC细胞,在Caski细胞中观察到较少的绿色荧光蛋白表达,而在HuVEC细胞中观察到较多的绿色荧光蛋白表达。结论survivin启动子能驱动shRNA在宫颈癌Caski细胞中特异性表达,而在正常HuVEC细胞中不表达。

Objective To construct pGenesil- 1 -EGFP- shRNA/survivin eukaryotic expression vector driven by the promoter of survivin gene and to test its expression in Caski cell line. Metheds shRNA eukaryotic expression vector pGenesil - 1 - EGFP - shRNA/survivin was constructed in which U6 promoter was replaced by survivin gene promoter, pGenesil -1- EGFP - shRNA/ survivin plasmid was transfected into Caski and HuVEC cells and the expression change of EGFP was observed in two cell lines. Results pGenesil - 1 - EGFP - shRNA/survivin vector was constructed successfully and transfected into Caski and HuVEC cell lines, after 72h, there was less green fluorescence in Caski cells, whereas there was more in HuVEC cells. Conchsions The survivin gene promoter can drive the specific expression of shRNA in Caski cell line, but not in normal HuVEC cell line.