人TRBP基因在HEK-293细胞中的表达
Expression of human TRBP gene in HEK-293 cells
李俊堂 1王立锋 1王芳 1许彦鸣 1杨安钢2
作者信息
- 1. 第四军医大学基础部生物化学与分子生物学教研室,陕西,西安,710033
- 2. 第四军医大学基础部生物化学与分子生物学教研室,陕西,西安,710033;第四军医大学基础部免疫学教研室,陕西,西安,710033
- 折叠
摘要
目的:构建携带人TRBP(TAR RNA结合蛋白)基因的真核表达载体,并将其在HEK-293细胞中表达.方法:用RT-PCR的方法扩增获得TRBP全长cDNA,然后克隆入pFLAG-CMV4真核表达载体.脂质体法瞬时转染HEK-293细胞,间接免疫荧光和Western Blot检测目的蛋白表达.结果:成功获得了全长的TRBP cDNA,构建了含人的TRBP cDNA的真核表达载体.转染HEK-293细胞后提取蛋白,经电泳观察到在Mr 46 000存在与目的蛋白分子质量相符的条带,该条带可被抗TRBP多克隆抗体及抗FLAG mAb特异性识别.结论:TRBP哺乳动物表达系统的建立,为进一步研究TRBP的生物学效应奠定了基础.
Abstract
AIM:To construct eukaryotic expression vector carrying human TRBP(TAR RNA-binding prorein)gene and express it in HEK-293 cells.METHODS:RT-PCR was used to obtain human full-length TRBP cDNA,Then it was cloned into the eukaryotic expression vector pFLAG-CMV4.After transfection into HEK-293 cells through lipofectamine 2000~(TM) transiently,the expression of target gene was detected by indirect immunofluorescence assay and Western Blot.RESULTS:The obtained human TRBP cDNA was consisitent with that in GenBank.The eukaryotic expression vector encoding TRBP was successfully constructed and expressed in HEK-293 cells.After eleetrophosis a protein band with molecular weight about 46 000 was found in the expression product,which is accordant with interest protein,and this band could be specifically recognized by anti-TRBP polyclonal antibody and anti-FLAG monoclona]antibody.CONCLUSION:The establishment of mammalian expression system of TRBP is of significance in further research for TRBP.
关键词
TAR/RNA/结合蛋白/pFLAG-CMV4/基因表达/HEK-293/细胞Key words
TAR RNA-binding prorein/pFLAG-CMV4/gene expression/HEK-293 cells引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据