人β-catenin启动子的克隆及活性分析  

Cloning human β-catenin gene promoter and analyzing its activity

作　　者：张菊[1] 关恒云[1] 张鹏举[1] 陈蔚文[1] 刘帅[2] 尚进[2] 唐传刚[2] 姜安丽[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学研究所,济南250012 [2]山东大学医学院05级六年制临床医学专业,济南250012

出　　处：《山东大学学报（医学版）》2009年第11期21-24,共4页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30870732);山东省自然科学基金资助课题(Y2005C03)

摘　　要：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.

关 键 词：Β-CATENIN 克隆 启动子 PC3细胞 

分 类 号：Q344.13[生物学—遗传学]