pEGFP-Egr-1表达质粒构建及其表达抑制HepG2细胞增殖  

Construction of pEGFP-Egr-1 Eukaryotic Expression Plasmid and Its Expression Inhibiting HepG2 Cell Proliferation

作　　者：黄心怡[1] 虞游[1] 金韵特 陈建明[1] 

机构地区：[1]杭州师范大学生命与环境科学学院,浙江杭州310036

出　　处：《杭州师范大学学报（自然科学版）》2016年第4期382-386,共5页Journal of Hangzhou Normal University(Natural Science Edition)

基　　金：杭州市社会发展自主申报项目(20160533B01);国家级大学生创新创业训练计划项目(201510346019)

摘　　要：将RT-PCR扩增的Egr-1基因连接到pMD-18载体上并测序鉴定,经测序正确的Egr-1基因亚克隆到绿色荧光蛋白真核表达载体pEGFP-C1上构建成重组质粒pEGFP-Egr-1.重组质粒pEGFP-Egr-1转染HepG2细胞后,运用荧光显微镜及荧光定量PCR技术分析其在HepG2细胞中的表达情况,并进一步运用WST-1法分析其表达对HepG2细胞增殖的影响.结果显示,重组质粒pEGFP-Egr-1构建正确,转染后在荧光显微镜下观察可见HepG2细胞发出绿色荧光信号,荧光定量PCR分析发现转染后HepG2细胞Egr-1基因mRNA水平明显升高.WST-1结果表明,与对照组相比,转染pEGFP-Egr-1质粒的HepG2细胞,其增殖速率明显下降.上述结果表明,pEGFP-Egr-1重组质粒构建成功,外来过表达Egr-1基因能够抑制HepG2细胞增殖.Egr-1 gene amplified by RT PCR was ligated into the pMI）-18 T-vector and confirmed by sequencing. Then the pMD-Egr-1 plasmid was digested and the cloned into the pEGFP-C1 to construct the pEGFP-Egr 1. The pEGFP-Egr-1 plasmid was transfected into HepG2 cells and its expression was analyzed by fluorescent microscope and qPCR. Meanwhile, the effect of pEGFP-Egr-1 expression on HepG2 cells proliferation was measured by WST-1 assay. It was shown that the recombinant plasmid pEGFP-Egr-1 was successfully constructed and the GFP signal was also observed; the result of qPCR showed the mRNA expression of Egr-1 gene was elevated after transfection of pEGFP-Egr-1 and the WST 1 result indicated the proliferation rate of HepG2 cells transfected with pEGFP-Egr-1 was decreased compared with the control cells transfected with pEGFP C1. Thus, the collected data suggested pEGFP-Egr-1 plasmid was successfully constructed and over-expression of Egr-1 gene could inhibit HepG2 cell proliferation.

关 键 词：EGR-1基因 pEGFP-Egr-1表达质粒 HEPG2细胞 细胞增殖 

分 类 号：Q253[生物学—细胞生物学] R363.1[医药卫生—病理学]