过表达KLF4重组质粒的构建及其在白血病K562细胞中的表达  

Construction of Recombinant Plasmid for Over-expressing KLF4 and Its Expression in K562 Cells

作　　者：李波[1] 胡海艳[1] 谭明贤[1] 

机构地区：[1]重庆三峡中心医院,中国重庆万州404000

出　　处：《生命科学研究》2017年第2期125-129,共5页Life Science Research

基　　金：重庆市万州区市政府计划项目(201303015)

摘　　要：为了构建过表达锌指转录因子Krüppel样因子4(Krüppel-like factor 4,KLF4)基因的重组质粒pEGFP-KLF4,观察其在白血病K562细胞中的表达,以RT-PCR法扩增人KLF4基因,构建pEGFP-KLF4重组质粒;经酶切及测序鉴定后,将pEGFP-KLF4质粒电穿孔转染白血病K562细胞(K562/pEGFP-KLF4组),设pEGFP-C1空质粒转染K562细胞(K562/pEGFP-C1组)及空白K562细胞作为对照,荧光显微镜观察EGFP-KLF4融合蛋白的表达情况,同时用抗生素G418筛选阳性克隆并扩大培养建立稳定过表达KLF4基因的K562细胞株,随后用RT-PCR检测3组K562细胞中KLF4的m RNA表达水平。实验结果显示,pEGFP-KLF4重组质粒构建成功。该质粒转染K562细胞24 h后能观察到较高强度的绿色荧光;经G418筛选出的阳性克隆扩大培养后建立了稳定过表达KLF4基因的K562细胞株;与对照组相比,K562/pEGFP-KLF4细胞组KLF4的m RNA表达水平明显升高,其过表达率为65.71%(P<0.05)。实验中构建的过表达KLF4基因的重组质粒pEGFP-KLF4能在K562细胞中高效表达,为进一步研究其在白血病中的作用奠定了基础。To construct the recombinant plasmid for over-expressing KrUppel-like factor 4 （KLF4） and to study its expression in K562 cells, human KLF4 gene was amplified by RT-PCR, and then cloned into pEGFP-C1 vector to construct pEGFP-KLF4. After confirmed by restriction analysis and sequencing, the recombinant plasmid was transfected into K562 cells by electroporation. The K562 cells with the recombinant plasmid were obtained as an experimental group （K562/pEGFP-KLF4）, and the cells with the vector （K562/pEGFP- C1） and the untreated cells were both used as control. The expression of pEGFP-KLF4 fusion protein in K562 cells was observed by fluorescence microscopy, and the G418-resistant clones of K562 cells were screened to establish stably transfected K562 cell line. RT-PCR was performed to detect the level of KLF4 mRNA in each group of cells. The results showed that the pEGFP-KLF4 plasmid was constructed successfully. Compared with K562/pEGFP-C1 group and K562 group, the experimental group had a significantly increased level of KLF4 mRNA, with over-expression ratio up to 65.71% （P〈0.05）. The recombinant plasmid proved to be able to over-express KLF4 and the expression level could be high in K562 cells, which lays a foundation for further study on the function of KLF4 over-expression in leukemia.

关 键 词：锌指转录因子Krüppel样因子4基因(KLF4) 过表达质粒 电穿孔转染 白血病 K562细胞 

分 类 号：Q786[生物学—分子生物学] R34[医药卫生—基础医学]