SRE元件报告质粒构建、转染和活性测定  

Construction,transfection and activity evaluation of luciferase reporter plasmid containing SRE element

作　　者：张弦[1] 王友平[1] 刘璐[1] 刘健[1] 

机构地区：[1]合肥工业大学生物与食品工程学院,安徽合肥230009

出　　处：《合肥工业大学学报（自然科学版）》2014年第1期110-113,共4页Journal of Hefei University of Technology：Natural Science

基　　金：安徽省自然科学基金资助项目(11040606M92)

摘　　要：固醇调节元件结合蛋白(SREBP)是机体调控脂质合成的重要转录因子,其作用靶标是所调控基因启动子区域的SRE元件,构建该元件报告质粒对于评价SREBP相关活性物质的作用机制具有重要意义。文章扩增或人工合成了人LDLr基因启动子区域SRE元件1次重复片段或3次重复片段,将其插入荧光素酶报告基因载体pGL3-basic中,构建了2种含有人SRE元件的荧光素酶报告质粒;将该报告质粒转染到人肝癌细胞HepG2中,胰岛素处理可以显著活化荧光素报告质粒,而25-羟基胆固醇和胆固醇结合处理可以强烈抑制。结果表明,所构建的质粒能成功地反映SREBP活性的改变,为体外快速检测SREBP活性相关调控物质提供了一个有效的平台。SREBP is an important transcription factor, which can regulate lipid synthesis. Its action target is SRE element in the promoter region of regulated gene. Therefore, the construction of lucifer- ase reporter plasmids containing SRE element is significant for the mechanism evaluation of SREBP associated bioactive compounds. In this paper, two kinds of luciferase reporter plasmids containing human SRE element were constructed by inserting cloned one repeated or synthetized three repeated SRE element fragments from human LDLr promoter into a luciferase reporter vector pGL3-basic. Next, these report plasmids were transfected into HepG2 cell line and tile activities of luciferase were characterized. The treatment of insulin could activate the luciferase reporter plasmids and the combi- nation of 25-hydroxycholesterol and cholesterol treatment could inhibit their activity. The results showed that the lueiferase reporter successfully reflected the alteration of SREBP activity, and it would be an effective and rapid platform for screening SREBP activity related substances.

关 键 词：SRE元件 荧光素酶报告质粒 活性测定 

分 类 号：Q786[生物学—分子生物学]