pEGFP-C2-MeCP2重组质粒的构建和表达  

Construction of recombinant pEGFP-C2-MeCP2 and its expression

作　　者：彭云云[1,2] 黄成[1,2] 马陶陶[1,2] 徐涛[1,2] 李琳[1,2] 陈晨[1,2] 李俊[1,2] 

机构地区：[1]安徽医科大学药学院,合肥230032 [2]安徽医科大学肝病研究所,合肥230032

出　　处：《安徽医科大学学报》2014年第9期1193-1197,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号81273526;81202978);新教师博士点基金(编号:20123420120001)

摘　　要：目的构建人类甲基化CpG结合蛋白(MeCP2)基因的真核表达载体pEGFP-C2-MeCP2,在非洲绿猴肾成纤维细胞(COS-7)中检测其表达及定位。方法从人肝癌细胞(HepG2)中获得cDNA,以其为模板扩增MeCP2全长编码基因,克隆到载体pEGFP-C2上。构建的重组质粒测序后转染至COS-7细胞中,荧光倒置显微镜观察绿色荧光蛋白的表达,检测MeCP2蛋白定位,Western blot法和RT-PCR法鉴定MeCP2蛋白和基因表达。结果成功构建pEGFP-C2-MeCP2重组质粒,酶切鉴定片段为1 461 bp。MeCP2蛋白及基因表达水平明显增加。细胞定位实验结果显示MeCP2蛋白定位于胞核。结论成功构建重组质粒pEGFP-C2-MeCP2,该质粒的构建为进行MeCP2功能研究提供了依据。Objective To construct the GFP-tagged eukaryotie expression vector of human MeCP2 gene and identi- fy its expression and localization in COS-7 cells. Methods Total RNA and cDNA were extracted from HepG2 cells, the MeCP2 coding sequence was amplified by PCR method and cloned into pEGFP-C2 vectors. After the target re- gion was sequenced, the recombinant vector was transfected into COS-7 cells, the expression and localization of pEGFP-C2-MeCP2 recombinant plasmid was monitored by immunofluorescence, PCR and Western blot. Results The recombinant vector was recombined successfully, the length of the fragment was about 1 461 bp. The protein and mRNA of MeCP2 were increased visibly. The nuclear fluorescence localization of MeCP2 protein was proved by immunofluorescence. Conclusion The eukaryotic expression vector of MeCP2 has been successfully constructed and expressed in COS-7 cells. The recombinant vector would be a useful material for MeCP2 function investigation.

关 键 词：MECP2 COS-7 基因表达 

分 类 号：R349.64[医药卫生—基础医学]