生命科学研究2018，Vol.22Issue(1)：19-25,50,8.DOI:10.16605/j.cnki.1007-7847.2018.01.004
人Dcp1a基因真核表达载体的构建及其在HeLa细胞中的定位
Construction of the Eukaryotic Expression Vector for Human Dcp1a Gene and Analysis of Its Cellular Localization in HeLa Cells
摘要
Abstract
Human mRNA decapping enzyme 1a (Dcp1a) is one of the important members of mRNA decay.Construction of its eukaryotic expression vector and analysis of its expression and cellular localization can provide a basis for further study of the function of Dcp1a.In this study,HeLa cDNA library was used as the template resource.The full-length cDNA sequence of Dcp1a was amplified by PCR method and subcloned into pmCherry-N1 vector.After transformation into E.coli DH5α,the positive clones were picked for plasmid extraction and identification by Xho Ⅰ/Sal Ⅰ double digestion and sequencing analysis.Then the recombinant plasmid was transfected into HeLa cells with VigoFect transfection reagent,and the expression of Dcp1a in HeLa cells was detected by RT-PCR and Western-blot.The cellular localization of Dcp1a was observed by confocal microscopy.The results demonstrated that the sequence of Dcp 1a gene was completely correct.The eukaryotic expression vector pmCherry-N1-Dcp1a was successfully constructed and Dcp 1a was overexpressed in HeLa cells.The confocal results showed that Dcp1a was localized in the cytoplasm and its overexpression in HeLa cells significantly induced the formation of processing bodies (P-bodies).These results lay the foundation for further study on the functions of Dcp1a and its correlation with P-bodies.关键词
人脱帽酶Dcp1a/载体构建/HeLa细胞/细胞定位/P小体Key words
human mRNA decapping enzyme 1a (Dcp1a)/vector construction/HeLa cells/cellular localization/processing body (P-body)分类
生物科学引用本文复制引用
赵雷,王申森,黄映辉,张丽娜..人Dcp1a基因真核表达载体的构建及其在HeLa细胞中的定位[J].生命科学研究,2018,22(1):19-25,50,8.基金项目
国家自然科学基金青年科学基金项目(81702272) （81702272）
北京工业大学基础研究基金资助项目(PXM2017_014204_500087) （PXM2017_014204_500087）
北京工业大学引进人才科研启动经费(01500051417010) （01500051417010）
