转录因子STAT5A启动子荧光素酶报告基因质粒的构建及鉴定

Construction and identification of luciferase reporter plasmids for the transcription factor STAT5A promoter

目的:利用分子克隆的方法构建转录因子STAT5A启动子荧光素酶报告基因质粒并鉴定其效应。方法:以乳腺癌MCF7细胞基因组DNA为模板,采用PCR方法扩增STAT5A基因的启动子序列,并将其克隆至荧光素酶报告基因质粒pGL3-Enhancer中,经过菌液扩增、酶切、测序等方法获得目的质粒,并通过双荧光报告基因实验进一步验证其功能。结果:构建的STAT5A启动子荧光素酶报告基因质粒pGL3-STAT5A-pro-luc-E序列正确,且具有转录活性。在双荧光报告基因实验中发现重组载体pGL3-STAT5A-pro-luc-E荧光素酶的相对活性约为阴性对照pGL3-Enhancer的8倍(P<0.01),而pGL3-STAT5A-pro-N-luc-E荧光素酶的相对活性约是阴性对照pGL3-Enhancer的6倍(P<0.01)。结论:本项目成功构建了转录因子STAT5A启动子荧光素酶报告基因质粒,为进一步研究STAT信号转导及转录激活蛋白信号通路提供了重要的研究工具。

OBJECTIVE:To construct and to evaluate activities of luciferase reporter plasmids for transcription factor STAT5A promoter.METHODS:The sequence for the promoter region in STAT5A gene was amplified by PCR using genomic DNA of the breast cancer cell line MCF7 as a template and cloned into luciferase reporter plasmid pGL3-Enhancer.The target plasmid was amplified by bacterial fluid,examined by enzyme digestion and direct sequencing.The function of target plasmid was further verified by dual luciferase reporter gene assay.RESULTS:It was found that the cloned sequence of STAT5A promoter region in target plasmid was correct,and the target plasmid had transcriptional activities.Dual-luciferase assays found that the relative activities of the recombinant vector pGL3-STAT5A-pro-luc-E luciferase were about 8 times that of the negative control pGL3-Enhancer(P<0.01),and the pGL3-STAT5A-pro-N-luc-E luciferase activities were about 6 times that of the negative control pGL3-Enhancer(P<0.01).CONCLUSION:A luciferase reporter plasmid for transcription factor STAT5A promoter was successfully constructed.The plasmids would provide an important research tool for further study of STAT signal transduction and the transcriptional activator protein signaling pathway.