小鼠adam10基因启动子克隆和双荧光素酶报告基因系统构建及鉴定  

Cloning of Mouse adam10 Gene Promoter and Construction and Identification of Dual Luciferase Reporter System

作　　者：陈伟[1,2] 陈翀[1] 张焕新[1] 曹江[1] 桑威[1] 吴庆运[1] 赵恺[1] 藏宇[1] 曾令宇[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏徐州221002 [2]南京医科大学第一临床学院血液科,江苏南京210029

出　　处：《中国实验血液学杂志》2012年第3期740-743,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30770915)

摘　　要：本研究旨在克隆小鼠adam10基因启动子,构建以adam10基因启动子为启动序列的双荧光素酶报告基因系统并分析其活性,为研究adam10基因转录调控提供有效工具。以BALB/c小鼠脑组织为模板,采用PCR方法克隆小鼠adam10基因启动子序列至pCR-Blunt载体,酶切后连接至荧光素酶报告质粒pGL4.10启动子区域,构建重组荧光素酶报告质粒pGL4.10-adam10,采用阳离子脂质体法与阳性对照质粒pGL4.74共转染真核细胞293FT,以离子霉素刺激为刺激组、DMSO为未加干预组,同时以不含启动子的pGL4.10与阳性对照质粒pGL4.74共转染组为阴性对照组,化学发光仪检测荧光强度及比例。结果表明,酶切及测序验证克隆的adam10启动子序列正确且无突变,构建的重组荧光素酶报告质粒pGL4.10-adam10与阳性对照质粒pGL4.74载体成功共转染293FT细胞,pGL4.10-adam10转染细胞后具有转录活性。离子霉素刺激组活性明显高于DMSO组,荧光比值约为DMSO组2倍(P<0.05),阴性对照组不具备转录活性。结论:成功克隆了adam10启动子并构建了重组荧光素酶报告质粒pGL4.10-adam10,离子霉素可增强adam10基因启动子转录活性。This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector,and to investigate its transcriptional activity.Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR.The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10.The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofetamine 2000.The activity of adam10 gene promoter was assayed by luciferase system.The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed.The method was optimized by changing ratio of two plasmids.Moreover,the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased.It is concluded that the dual luciferase reporter system is successfully established,which is useful in bioluminescence imaging technology in vitro.The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.

关 键 词：解聚素金属蛋白酶 聚合酶链反应 报告质粒 293FT细胞 

分 类 号：Q782[生物学—分子生物学] R733.1[医药卫生—肿瘤]