人β-catenin启动子的克隆及活性分析

Cloning human β-catenin gene promoter and analyzing its activity

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。

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 genomic DNA as a template.Its promoter activity was determined with dual-luciferase reporter assay after it had been cloned into a pGL3-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（Ml/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 pGL3-control co-transfection with pRL-TK,206.31 fold higher than that of pGL3-basic co-transfection with pRL-TK and 21.38 fold higher than that of pGL3-promoter cotransfection with pRL-TK.Conclusion The cloned 1.8 kb fragment upstream of the β-catenin gene presented strong promoter activity.