人ADAM17启动子荧光素酶报告质粒的构建与鉴定

Construction and identification of luciferase reporter plasmid for human ADAM17 promoter

目的构建人去整合素-金属蛋白酶17(ADAM17)启动子的荧光素酶报告质粒,对不同位点进行定点突变,分析其转录活性。方法通过生物信息学分析ADAM17启动子区域的叉头转录因子M1(FoxM1)结合位点,以人胃癌细胞系MGC-803基因组为模板,PCR扩增ADAM17基因启动子-1 365^+51片段,连接到荧光素酶报告基因载体pGL3-basic中;对FoxM1结合的3个位点进行定点突变;转染至293T细胞后用双荧光素酶报告系统分析其转录活性。结果测序结果显示,ADAM17启动子荧光素酶报告基因及其突变体均构建成功;与pGL3-basic组相比,pGL3-ADAM17(-1 365^+51)组介导的荧光素酶活性明显升高(P<0.05);与对照组相比,过表达FoxM1组介导的荧光素酶活性明显升高(P<0.05),且pGL3-ADAM17(-1 365^+51)组高于突变组(P<0.05)。结论成功构建了人ADAM17启动子荧光素酶报告基因及其突变体,初步验证了ADAM17受转录因子FoxM1的转录调控。

Objective To construct a luciferase reporter plasmid of human ADAM17 gene promoter and mutate specific sites,and to identify their biological activity.Methods FoxM1 binding sites on ADAM17 promoter were analyzed by bioinformatics.The target fragment was amplified with PCR by using genomic DNA of human gastric cancer cell line MGC-803 as template,and ligated to luciferase reporter vector pGL3-basic;the three FoxM1 binding sites were mutated on the basis of recombinant plasmid;these plasmids were transferred into 293T cells and the promoter activity was determined by double luciferase reporter gene detection system.Results The sequencing results showed that the ADAM17 promoter fluorescent reporter gene and its mutants were successfully constructed.Compared with the pGL3-basic group,the luciferase activity mediated by pGL3-ADAM17(-1 365^+51)group was significantly increased(P<0.05);Compared with the control group,the luciferase activity mediated by over-expressing FoxM1 significantly increased(P<0.05),and the pGL3-ADAM17(-1 365^+51)group was higher than that of the three mutation groups(P<0.05).Conclusions The human ADAM17 promoter luciferase reporter gene and its mutants are successfully constructed,and the transcriptional regulation of ADAM17 by FoxM1 is preliminarily verified.