含hITF启动子的荧光素酶报告质粒的构建及活性检测

Construction of a luciferase reporter vector containing hITF promoter

目的构建含有人肠三叶因子(intestinal trefoil factor,hITF)启动子的荧光素酶表达载体,并检测启动子活性。方法采用PCR技术从LS-174T细胞基因组DNA中扩增出长约2.5kb的含hITF基因启动子片段(-2331/+53),而后在此基础上构建了10个不同长度的启动子序列至pGL-3basic荧光素酶表达载体,然后将这些重组质粒转染HEK-293细胞并在48h后检测其荧光素酶活性。结果插入的hITF启动子片段测序结果显示正确,-500/+53,-400/+53和-300/+53片段活性介于0.023～0.026之间,-200/+53,-100/+53和-50/+53活性介于0.0032～0.0042之间,明确了最小活性功能域位于-300/+53区域,-300/-200区域对hITF转录起始起关键性作用。结论成功构建了含hITF启动子的萤火虫荧光素酶报告基因质粒,鉴定出人肠三叶因子(hITF)启动子活性位于-300/-200区域。

Objective To construct a luciferase reporter vector containing human intestinal trefoil factor （hITF） promoter and to evaluate its activity in LS-174T and HEK-293 cells.Methods A 2.5 kb fragment spanning the region-2 331 to ＋53 of hITF gene was amplified from genomic DNA extracted from LS-174T by PCR.Then 10 fragments containing different region of the promoter were substantially inserted into promoter-less pGL-3 basic luciferase reporter vector,and the obtained vectors were transfected into HEK-293 cells.In 48 h after the transfection,the cells were lysed and the luciferase activity was measured.Results The inserted hITF promoter was verified by DNA sequencing.The promoter activity of-500/＋53,-400/＋53 and-300/＋53 ranged from 0.023 to 0.026,while that of-200/＋53,-100/＋53 and-50/＋53 from 0.003 2 to 0.004 2.The minimal functional promoter was located in the-300/＋53,and there were some potential active elements.The promoter region of-300/-200 played a vital role in transcription initiation of hITF.Conclusion The recombinant plasmid containing luciferase reporter gene driven by hITF promoter is constructed successfully.The region spanning-300/-200 plays an important role in facilitating the initiation of transcription.