小鼠TIE2基因启动子的克隆及转录活性分析

Cloning and Analyzing the Promoter of Mouse TIE2 Gene

克隆小鼠TIE2基因的启动子并分析其转录活性。设计并合成引物,以小鼠肝脏组织DNA为模板,巢式PCR扩增小鼠TIE2基因启动子区。将扩增获得的系列截短片段克隆入荧光素酶(Luc)报告基因表达载体pGL3-Basic中,构建系列启动子区转录活性报告质粒pGL3-TIE2-Luc。报告质粒与内参质粒共转染SVEC4-10、NIH3T3、HUVEC及NIT-1细胞系,48 h后收获细胞检测双荧光素酶的表达情况。构建的pGL3-TIE2-Luc系列报告质粒经过酶切鉴定及DNA测序分析都显示正确;转染4种细胞系后进行双荧光素酶活性检测的结果表明,TIE2基因启动子区域(-2056-+1)具有较强的转录活性。成功构建小鼠TIE2基因启动子报告质粒,证实TIE2基因上游区域(-2056-+1)具有较强的启动子活性。

It was to clone the promoter of mouse TIE2 gene and analyze the transcription activity.Nest PCRs were used to obtain DNA fragments that contained the promoter of TIE2 gene using genome DNA prepared from mouse liver as the templates.Reporter plasmids pGL3TIE2-Luc contained 5’serial truncated promoter were constructed.After the reporter plasmids were transiently transfected into SVEC410,NIH3T3,HUVEC and NIT-1 cells,the luciferase activity were tested.Result showed that mouse TIE2 promotor fragments were correctly amplified and the reporter plasmids were successfully constructed;Dual luciferase assays of the constructed reporter plasmids in four cell lines showed that the promoter region（-2056-＋1）had strong transcriptional activity.Therefore,the promoter of mouse TIE2 gene was successfully cloned.It confirmed that TIE2 promoter region（-2056-＋1）had strong transcriptional activity in cell lines.