大鼠GLUT3基因启动子区荧光素酶报告基因载体的构建与鉴定

目的:克隆大鼠葡萄糖转运体3(glucose transporter 3,GLUT3)基因启动子区,并构建其萤火虫荧光素酶报告基因载体。方法:在对大鼠GLUT3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的方法从大鼠基因组中扩增出GLUT3基因5′侧翼区-1037～155bp段长为1 292bp的启动子区(以翻译起始点ATG为+1),再用定向克隆的方法将这一启动子区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(pGL3-basic)中,构建出包含大鼠GLUT3基因启动子区的萤火虫荧光素酶报告基因载体(pGL3-GLUT3),电泳与测序鉴定,最后再将pGL 3-GLUT3与内参pRL-TK用脂质体转染的方法瞬时共转染PC12和原代培养的神经元中,通过双荧光素酶报告基因检测系统鉴定pGL 3-GLUT3的启动子活性,并用独立样本t检验方法进行统计分析。对照组共转染pGL3-basic与内参pRL-TK。结果:构建出荧光素酶报告基因载体pGL3-GLUT3。与转染空质粒pGL3-basic组相比,原代神经细胞中转染pGL3-GLUT3组荧光素酶活性升高(5.182 9±0.264 8 vs 2.893 1±0.775 4,P=0.008),在PC12细胞中转染pGL3-GLUT3组荧光素酶活性也升高(2.797 7±0.512 0 vs 1.179 8±0.312 5,P=0.010)。结论:成功克隆GLUT3基因启动子区,并构建出包含GLUT3基因启动子片段的荧光素酶报告基因载体,并且在PC12和原代培养的神经元中pGL 3-GLUT3可以表现出启动子活性。这为后续大鼠GLUT3基因转录调控研究提供研究素材。

Constructing and identifying of the firefly luciferase reporter gene vector pGL3-GLUT3

Objective:To clone the rat GLUT3 promoter,construct and identify the firefly luciferase reporter gene vector pGL3-GLUT3.Methods:The promoter sequences of rat GLUT3 gene was obtained by PCR method.The product of PCR was inserted into pGL3-basic vector by T4 DNA ligase after double digestion with Kpn Ⅰand XhoⅠ.The constructed vector pGL3-GLUT3 was transferred into DH5a E.coli.The positive clone was identified by double digestion with Kpn Ⅰand Xho Ⅰand DNA sequencing.Finally,pGL3-GLUT3 was contransferred with pRL-TK into PC12 cells and primary culture neurons by lipofectamine 2000 to identify its promoter function.The control group was contransferred with pRL-TK and pGL3-basic.Results:The luciferase activity in primary culture neurons group was higher than that of control group(5.1829±0.2648 vs 2.8931±0.7754,P=0.008).The luciferase activity in PC12 cells group was 2.7977±0.5120,and that of its control was 1.1798±0.3125,P=0.010.Conclusions:A 1292 bp rat GLUT3 promoter was successfully cloned and the firefly luciferase reporter gene vector pGL3-GLUT3 was well constructed.In PC12 cells and primary culture neurons,pGL3-GLUT3 has the promoter function.