小鼠TIE2基因启动子的克隆及其转录活性分析.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 2012年第11期 小鼠 TIE2 基因启动子的克隆及转录活性分析 陆俊佳  袁志刚  许淑茹  廖明  赖青鸟  畅荣妮  袁广之  侯伟  舒伟 （广西医科大学细胞生物学与遗传学教研室，南宁 530021） 摘 要 ： 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。 关键词 ： TIE2 启动子 双荧光素酶活性检测 Cloning and Analyzing the Promoter of Mouse TIE2 Gene Lu Junjia Yuan Zhigang Xu Shuru Liao Ming Lai Qingniao Chang Rongni Yuan Guangzhi Hou Wei Shu Wei （Dep artment of Cell Biology and Genetics ，Guangxi Medical University ，Nanning 530021 ） Abstract:  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 pGL3- TIE2-Luc contained 5 ’ serial truncated promoter were constructed. After the reporter plasmids were transiently transfected into SVEC4- 10, 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