人IGF1R启动子克隆及荧光素酶报告基因载体构建

Cloning of Human IGFIR Promoter and Construction of the Promoter Luciferase Reporter Gene Vectors

目的克隆人1GF1R基因启动子序列，构建IGF1R基因启动子荧光素酶报告基因载体。方法运用PCR方法从HeLa细胞基因组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IGF1R基因启动子报告基因载体；将重组质粒转染至HeLa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800bp左右IGF1R基因启动子片段；双酶切及测序鉴定重组体构建正确；转染HeLa细胞后经双荧光素酶报告基因检测确定重组体有启动子活性。结论成功构建了IGF1R启动子报告基因载体，在细胞系中进行初步的活性分析得知所克隆的IGF1R基因调控序列内包含启动子活性区域。

Objective To clone the human IGF1R gene promoter, and to construct the lucifer- ase reporter gene vector containing the IGF1R gene promoter sequence. Methods The IGF1R pro- moter fragment was amplified from genomic DNA of HeLa cells by PCR. After digestion with restriction enzymes, the IGF1R promoter was cloned into the luciferase reporter vectors. Then the recombinant vector was transiently transfected into HeLa cells with pRL-TK. The activity of luciferase was detected 48 hours later. Results The IGF1R gene promoter, a fragment about 800 bp, was amplified by PCR. Double digestion and sequencing revealed that the recombined plasmid was correctly constructed. The dual-luciferase reporter assay showed that the recombinant vector had a promoter activity. Conclusion The human IGF1R promoter luciferase reporter gene vector was successfully constructed. The cloned IGF1R gene fragment was demonstrated to contain the cis-regulatory elements.