重组质粒pmCherry-WT-hERG和pEGFP-E637K-hERG的构建及其在细胞内的表达和定位  

Construction of recombinant plasmids pmCherry-WT-hERG and pEGFP-E637K-hERG and their expression and localization in cells

作　　者：黄晓燕[1] 杨曦[1] 廉姜芳[1] 

机构地区：[1]宁波市医疗中心李惠利医院器官移植重点实验室,浙江宁波315040

出　　处：《中国卫生检验杂志》2015年第8期1216-1219,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金资助课题(81370207);宁波市自然科学基金资助课题(2014A610276)

摘　　要：目的分别构建先天性长QT综合征(LQTS)相关HERG基因和E637K突变基因与红色和绿色荧光蛋白基因的融合表达载体pmCherry-WT-hERG和pEGFP-E637K-bERG,观察其在细胞内的表达和定位情况。方法将克隆在pcDNA3上的HERG和E637K片段分别亚克隆到红色荧光蛋白载体pmCherry-C2和绿色荧光蛋白载体pEGFP-C1上,转染HEK293T细胞,24 h后利用western blot技术和荧光显微镜观察重组质粒蛋白表达和细胞内定位情况。结果重组质粒经酶切、PCR和测序鉴定正确无误。转染pmCherry-WT-hERG野生型质粒的细胞可以检测到135 kD和155 kD 2条蛋白质条带,而转染pEGFP-E637K-bERG突变型质粒的细胞仅显示1条135 kD蛋白质条带,155 kD处条带缺失。融合蛋白发出的红色和绿色荧光表明,突变型蛋白分布于胞浆中,而野生型蛋白主要位于胞膜上。结论成功构建了pmCherry-WT-hERG和pEGFP-E637K-hERG不同荧光蛋白融合表达载体,并在真核细胞中得到有效表达,为LQTS突变基因的进一步功能研究奠定了基础。Objective To construct pmCherry - WT - hERG and pEGFP - E637K - hERG fusion expression vectors of congeni- tal long QT syndrome related HERG gene and E637K mutation fused with red fluorescent protein or enhanced green fluorescent protein, and observe their expression and localization in HEK293T cells. Methods The PCR sequences of HERG and E637K in pcDNA3 vectors were sub cloned into pmCherry - C2 or pEGFP - C1 vectors, followed by transfected into HEK293T ceils for 24 hours. The expression and intracellular localization of these recombinant plasmids were detected by western blot and fluores- cence microscope. Results The two recombinant plasmids were verified by enzyme digestion, PCR and sequencing. The pm- Cherry - WT - hERG channel protein appeared in two protein bands, a lower band at 135 kD and an upper band at 155 kD. In contrast, the pEGFP - E637K - hERG resulted in a band only at 135 kD and the 155 kD protein band was absent. With fluores- cence microscope observation,the protein of pEGFP- E637K- hERG mutation was expressed in cytoplasm while the wild type pmCherry - WT - hERG was expressed mainly on cellular membrane. Conclusion The fusion expression vectors of pmCherry - WT- hERG and pEGFP -E637K- hERG are successfully constructed and expressed efficiently in eukaryotic cells, which pro- viding an important tool for the further study on functions of mutation.

关 键 词：先天性长QT综合征 HERG基因 E637K突变体 真核表达载体 

分 类 号：R541.1[医药卫生—心血管疾病]