MDR1启动子/荧光素酶质粒的构建及其在朊蛋白高表达胃癌细胞中的活性检测

目的:构建含MDR1基因启动子的荧光素酶报告基因质粒,并检测其在朊蛋白高表达胃癌细胞系中的活性表达。方法:PCR克隆人MDRl基因启动子片段,通过亚克隆将启动子分别插入到pMD18-T载体和荧光素酶报告基因pGL3-Enhancer载体中,建立含MDR1启动子的荧光素酶报告基因质粒pGL-MDR1,并经测序及酶切确定扩增序列;脂质体基因转染法将pGL-MDR1转染入朊蛋白高表达胃癌细胞系SGC7901-PrP,并测定其荧光素酶活性。结果:PCR克隆出MDR1启动子经DNA测序证实序列正确,pGL-MDR1转染入朊蛋白高表达胃癌细胞系的荧光素酶活性,较转染入pcDNA3.1空载体细胞系相比升高3~5倍。结论:成功构建含MDRl启动子的荧光素酶报告基因质粒;上调朊蛋白表达可激活MDR1的转录活性。

Construction of luciferase reporter gene vector containing MDRI promoter gene and MDR1 promoter activity in PrPc overexpressed gastric cancer cells

Objective: To construct the luciferase reporter vector containing MDRl promoter gene and detect the Luc activity in PrPc overexpressed gastric cancer cells.Methods:The promoter of the human MDRl gene was amplified from human genomic DNA by PCR;then it was subcloned into pMD18-T and then pGL3-enhancer vector.The plasmid was determined by restriction enzyme digestion and DNA sequencing;then it was transfected into PrPc overexpressed gastric cancer cell line SGC7901-PrP by Lipofectamine 2000.At last,the luc activity was detected.Results:After correctly constructed the pGL3-MDR1 promoter plasmid,the luc activity in PrPc overexpressed gastric cancer cells were found to be 3~5 fold greater than those of empty vector control.Conclusion: The luciferase reporter gene vector containing MDRl promoter was successfully constructed and upregulation of PrPc expression could lead to the activation of MDR1 promoter in gastric cancer cells.