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

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1 308 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 (pGL4.10) 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 (pGL4.10-TBX21pr-CNS)，电泳与测序鉴定，最后再将pGL4.10-TBX21pr-CNS与内参pRL-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pGL4.10-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pGL4.10与内参pRL-TK。结果表明，成功构建出荧光素酶报告基因重组质粒pGL4.10-TBX21pr-CNS。与转染空质粒pRL-TK组相比，293T细胞 (P＝0.012 2) 和Jurkat细胞 (P＝0.002 2) 中转染pGL4.10-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pGL4.10-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。

Abstract:

The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-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 pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with KpnⅠand SfiⅠand DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T 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 pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.