人β1整合素近端和远端启动子报告基因载体的构建和鉴定及启动活性分析  

Construction and identification of integrin β1 recombinants containing the proximal and distal promoter and detection of their promoter activity

作　　者：陈卫[1] 苏踊跃[1] 梁光萍[1] 陈建[1] 张曼辉 罗向东[1] 

机构地区：[1]第三军医大学西南医院烧伤研究所创伤烧伤与复合伤国家重点实验室,重庆400038 [2]解放军第二六三医院超声科,北京101149

出　　处：《医学研究生学报》2007年第2期135-137,141,共4页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:10332060);国家重点基础研究发展规划资助项目(批准号:2005CB522601)

摘　　要：目的:克隆整合素β1近端启动子和远端启动子基因序列,构建并鉴定整合素β1启动子调控的荧光素酶报告基因载体pGL3-261和pGL3-1442。方法:以本室构建的含整合素β1全长启动子基因序列(1756 bp)的重组载体pGL3-β1为模板,用含有酶切位点的特异性引物扩增出整合素β1近端和远端启动子基因序列,克隆至荧光素酶表达载体pGL3-basic,构建含正确目的基因的表达载体pGL3-261和pGL3-1442,经NheⅠ、XhoⅠ酶切、聚合酶链式反应(PCR)扩增及测序鉴定;并用pGL3-261和pGL3-1442质粒与pGL3-β1质粒同时转染人表皮细胞株HaCaT进行活性分析。结果:酶切及序列测定表明,克隆获得的近端启动子261bp和远端启动子1442bp与GenBank DNA序列数据库对比分析序列一致,且插入方向正确;此三种质粒都有明显的启动子活性,远端启动子活性与全长启动子活性无显著差异,但明显高于近端启动子活性。结论:成功构建了整合素β1近端和远端启动子基因序列荧光素酶报告基因载体,整合素β1远端启动子在HaCaT细胞中起主要作用。为下一步研究人整合素β1启动子核心区、基因表达调控机制及其信号转导通路等奠定基础。Objective ：To clone the proximal and distal promoter of integrin β1 gene, and to construct and identify integrin β1 gene promoter regulated luciferase reporter vector pGL3-261 and pGL3-1442. Methods： Recombinant of pGL3-β1 vector including the proximal promoter sequence and the distal promoter sequence of integrin β1 gene were used as template in polymerase chain reaction（PCR） for amplification of its promoter sequence. The PCR product was directly cloned into the luciferase reporter vector pGL3 - basic, the products were identified by DNA sequencing and restriction enzymes digestion, and the three vectors containing integrin β1 full length promoter, the proximal and distal promoter were transfected into HaCaT cells. The luciferase activity of the cells were analyzed and compared after transfeeton. Results ： Restriction enzymes digestion and DNA sequencing confirmed that the sequence of the recombinant were identical to that in GeneBank, and the segment was inserted in right direction. The activity of the three vectors was significantly higher than that in pGL3-basic vectors. The activity of the distal promoter was significantly higher than those of proximal and distal promoters. There was no obvious difference of the luciferase activity between the distal promoter and the full length promoter. Conclusion ：The Iuciferase expression vectors of pGL3-261 and pGL3-1442 containing the proximal and distal promoter sequence of Integrin β1 gene is constructed successfully.

关 键 词：整合素Β1 远端启动子 近端启动子 载体 

分 类 号：Q782[生物学—分子生物学]