人β catenin启动子的克隆及活性分析

目的克隆β catenin基因5′上游1.8?kb启动子，构建启动子 荧光素酶报告基因载体pGL3 1.8?kb，测定其启动子活性，为进一步研究β catenin基因表达调控机制奠定基础。方法采用PCR方法从人基因组DNA中扩增β catenin基因5′上游1.8?kb片段并构建到荧光素酶报道基因pGL3 basic载体中，与内参照质粒pRL Tk共转染前列腺癌PC3细胞，通过双荧光素酶活性检验测定其启动子活性。结果PCR扩增的1.8?kb片段经测序正确无误；pGL3 1.8?kb转染PC3细胞48?h后，双荧光素酶活性测定启动子活性（M1／M2）为11.71，是pGL3 control活性的2.43倍，为pGL3 basic活性的206.31倍,为pGL3 promoter活性的21.38倍。结论克隆的β catenin基因5′上游1.8?kb片段具有较强的启动子活性。

Cloning human β catenin gene promoter and analyzing its activity

Objective To clone a 1. 8 kb fragment upstream of the βcatenin gene and assay its promoter activity. Methods A 1.8 kb fragment upstream of the βcatenin gene was amplified by PCR using human genomie DNA as a template. Its promoter ac-tivity was determined with dual-luciferase reporter assay after it had been cloned into a pGL_3-basic vector and transfected into PC3 cells. Results The sequence of the 1.8 kb fragment proved to be correct by DNA sequencing. Dual-luciferase reporter assay (M1/M2) was 11.71 at 48 h after PGL3-1.8 kb was co-transfected with pRL-TK into prostate cancer cell PC3 which was about 2. 43-fold higher than that of pGL_3-control co-transfection with pRL-TK, 206.31 fold higher than that of pGL_3-basic co-transfection with pRL-TK and 21.38 fold higher than that of pGL_3-promoter cotransfection with pRL-TK. Conclusion The cloned 1.8 kb fragment upstream of the β-catenin gene presented strong promoter activity.