论文部分内容阅读
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。
The mouse TIE2 gene promoter was cloned and analyzed for transcriptional activity. Primers were designed and synthesized. The mouse liver tissue DNA was used as a template to amplify the mouse TIE2 gene promoter region by nested PCR. A series of truncated fragments were cloned into the luciferase (Luc) reporter gene expression vector pGL3-Basic to construct a series of promoter region transcriptional activity reporter plasmid pGL3-TIE2-Luc. The reporter plasmid and the reference plasmid were co-transfected into SVEC4-10, NIH3T3, HUVEC and NIT-1 cell lines, and the expression of dual luciferase was detected 48 h later. The constructed pGL3-TIE2-Luc reporter plasmids were verified by restriction analysis and DNA sequencing. The results of dual luciferase assay after transfection of four cell lines showed that the promoter region of TIE2 gene (-2056- + 1) has a strong transcriptional activity. The mouse TIE2 gene promoter reporter plasmid was successfully constructed and confirmed that the upstream region of TIE2 gene (-2056- + 1) has strong promoter activity.