小鼠T-bet启动子荧光素酶报告基因载体的构建及活性鉴定  

Construction and identification of luciferase reporter gene containing mouse T-bet promoter

作　　者：徐惠娟[1] 周守标[1] 

机构地区：[1]安徽师范大学生命科学学院安徽省重要生物资源保护与利用研究省级重点实验室,安徽芜湖241000

出　　处：《生物工程学报》2014年第11期1733-1741,共9页Chinese Journal of Biotechnology

基　　金：安徽省自然科学基金(No.11040606M77);安徽省高校自然科学基金重点项目(No.KJ2011A129)资助~~

摘　　要：为了研究T-bet在T细胞中的转录调控机制,并研究其在多发性硬化症中的信号通路,本研究构建小鼠TBX21(编码T-bet)基因启动子区和增强子区萤火虫荧光素酶报告基因载体。在对小鼠TBX21基因5?侧翼区进行详尽生物信息学特征分析后,设计相应引物,用PCR的方法从小鼠基因组中扩增出TBX21基因5?侧翼区–1 000 bp-28 bp片段长为1 028 bp的启动子区(以翻译起始点ATG为+1)和–3 308 bp-–2 000 bp片段长为1308 bp的非编码区保守序列(No-coding conserved sequence,CNS),再用定向克隆的方法将这两个片段定向重组入专门用于启动子活性研究的萤火虫荧光素酶报告基因载体(p GL4.10)中,构建出包含小鼠TBX21基因启动子区和CNS区的萤火虫荧光素酶报告基因载体(p GL4.10-TBX21pr-CNS),电泳与测序鉴定,最后再将p GL4.10-TBX21pr-CNS与内参p RL-TK用lipofectamine 2000共转染293T细胞和Jurkat细胞中,通过双荧光素酶报告基因检测系统鉴定p GL4.10-TBX21pr-CNS的启动子和增强子活性,并用独立样本t检验方法进行统计分析。对照组共转染p GL4.10与内参p RL-TK。结果表明,成功构建出荧光素酶报告基因重组质粒p GL4.10-TBX21pr-CNS。与转染空质粒p RL-TK组相比,293T细胞(P=0.012 2)和Jurkat细胞(P=0.002 2)中转染p GL4.10-TBX21pr-CNS组荧光素酶活性升高。研究结果表明在293T细胞和Jurkat细胞中p GL4.10-TBX21pr-CNS可以表现出启动子活性,为后续小鼠T-bet转录调控研究提供了基本材料。The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid p GL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into p GL4.10. The recombinant plasmid p GL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with KpnⅠ and SfiⅠand DNA sequencing. Finally, p GL4.10-TBX21pr-CNS was cotransfected with p RL-TK into 293 T cells and Jurkat cells, p RL-TK and p GL4.10 as a control. The luciferase activity in 293 T cells（P=0.012 2） and Jurkat cells（P=0.002 2） was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid p GL4.10-TBX21pr-CNS was constructed. In 293 T cells and Jurkat cells, p GL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.

关 键 词：T-BET 启动子 荧光素酶 报告基因 转录调控 

分 类 号：Q78[生物学—分子生物学]