人同源盒基因Nkx3.1启动子的克隆及活性测定  

Cloning of human Nkx3.1 gene promoter and assay of its promoter activity

作　　者：姜安丽[1] 王咏梅[2] 张建业[1] 胡晓燕[1] 贺美兰[1] 刘志芳[1] 

机构地区：[1]山东大学医学院生物化学教研室,山东济南250012 [2]山东济南卫生学校,山东济南250000

出　　处：《山东大学学报（医学版）》2004年第3期274-276,283,共4页Journal of Shandong University:Health Sciences

摘　　要：目的:克隆Nkx3.1基因5＇上游1.06 kb片段,构建pGL3-1.06 kb载体,测定其启动子活性?方法:采用PCR方法从人基因组DNA中扩增Nkx3.1基因5＇上游1.06 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LNCaP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1.06 kb片段经测序正确无误;pGL3-1.06 kb转染LNCaP细胞48 h后,双荧光素酶活性测定M1/M2=2.7, 为pGL3-control 活性的1.5倍,为pGL3-basic活性的50倍?结论:克隆的人Nkx3.1基因5＇上游1.06 kb片段具有较强的启动子活性?Objective: To clone 1.06kb fragment upstream of Nkx3.1 gene and assay its promoter activity. Methods: 1.06kb fragment upstream of Nkx3.1 gene was amplified by PCR using human genomic DNA as template. Its promoter activity was determined with dual-luciferase reporter assay after it had been cloned into pGL3-basic vector and transfected into LNCaP cells. Results: The sequence of the 1.06kb fragment proved to be correct by DNA sequencing. Dual-luciferase reporter assay (M1/M2) was 2.7 at 48 hours after pGL3-1.06kb was cotransfected with pRL-TK into prostate cancer cell LNCaP, which was about 1.5-fold higher than that of pGL3-control cotransfection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. Conclusion: Cloned 1.06kb fragment upstream of Nkx3.1 gene presented a strong promoter activity. [

关 键 词：基因 NKX3.1 克隆 分子 LNCAP细胞 遗传载体 

分 类 号：Q344.14[生物学—遗传学]