肠三叶因子启动子荧光素酶报告基因载体的构建及活性分析

Construction and Analysis of Luciferase Reporter Vectors of ITF Gene Promoter

目的:克隆肠三叶因子(ITF)启动子序列,构建ITF启动子荧光素酶报告基因载体,并检测启动子活性。方法:运用PCR方法从人全血基因组DNA中获得目的基因;双酶切后连接到荧光素酶报告基因载体上,构建不同长度的ITF启动子报告基因载体(-100--1 826bp);将重组质粒转染至HEK293细胞和LS174细胞48h后,采用双荧光素酶报告基因系统检测其启动子活性。结果:PCR扩增出不同长度的ITF启动子片段;双酶切及测序鉴定重组体构建正确;转染HEK293细胞和LS174细胞后检测启动子活性,100和200bp启动子活性较低,而300至1 826bp启动子活性较强。结论:成功构建了ITF启动子报告基因载体,具有活性的启动子最小长度为300bp。

Objective： To clone intestinal trefoil factor （ITF） gene promoter, construct its luciferase reporter vector and analyze its transcriptional activity. Methods. The promoter sequence of the ITF gene was cloned from human whole blood genomic DNA by PCR. Truncated promoter fragments with different lengths were cloned and inserted into the pGL3-Basic vector to construct recombinant plasmids. The recombinant plasmids were transiently transfected into HEK293 cells and LS174T cells, and then the activity of luciferase was detected. Results： ITF promoter fragments with different lengths were amplified by PCR. Restriction endonuclease analysis and DNA sequencing confirmed the recombinant plasmids were constructed successfully. The above plasmids were transfected into HEK 293 cells and LS 174 cells, and the activities of the promoter （100, 200 bp） were lower and the activities （300 to 1 826 bp） were higher. Conclusion： ITF promoter luciferase reporter gene vectors were constructed successfully and the minimal sequence required to maintain basic transcriptional activity was 300 bp.