NLS-RARα与ISCA1相互作用在哺乳动物细胞中的验证  

Identification of interaction between ISCA1 and NLS-RARα by co-immunoprecipitation in mammalian cells

作　　者：王东生[1,2] 王春光[1] 王翀 曹炬[1] 张国元[2] 王勇[2] 钟梁[1] 刘北忠[1] 

机构地区：[1]重庆医科大学临床检验诊断学教育部重点实验室,重庆400016 [2]川北医学院附属医院检验科,四川南充637000

出　　处：《第四军医大学学报》2009年第6期489-492,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30300449);国家中医药管理局(02-03ZP52);重庆医科大学基金(XBYB2007108;XBYB2007104)

摘　　要：目的:利用免疫共沉淀和蛋白质印记技术验证铁硫簇组装蛋白1(ISCA1)与带核定位信号的维甲酸受体α(NLS-RARα)蛋白间的相互作用.方法:构建含融合蛋白的真核表达载体pCMV-HA-NLS-RARα和pCMV-Myc-ISCA1,酶切及测序鉴定正确后,转染人胚肾293细胞,利用免疫共沉淀和蛋白质印记技术进一步证实二者之间的相互作用.结果:真核表达载体成功构建并经测序鉴定,转染293细胞,抗HA多克隆抗体沉淀HA-NLS-RARα相互作用蛋白复合物后,用抗Myc mAb进行蛋白印记检测,可以检测到Myc-ISCA1蛋白的表达.结论:分别成功构建了含HA-NLS-RARα与Myc-ISCA1融合蛋白的真核表达载体,并利用免疫共沉淀和蛋白质印记技术在体外证实了NLS-RARα与ISCA1之间存在相互作用.AIM： To verify the interaction between ISCAI and NLS-RARα by co-immunoprecipitation and Western blotting. METHODS： The eukaryotic expression vector pCMV-HA-NLS- RARer and pCMV-Myc-ISCA1 were constructed, identified and then transfected into human embryo kidney 293 cells. Co-immunoprecipitation and Western blotting were used to investigate the interaction between ISCA1 and NLS-RARα. RESULTS： After being verified, eukaryotic expression vectors were co-transfected into HEK 293 cells, HA-NLS-RARα protein was then immunoprecipitated by anti-HA polyclonal antibody, and Myc-ISCA1 protein was detected by western blotting with anti-Myc monoclonal antibody from the immunoprecipitared complex. CONCLUSION： Eukary- otic expression vectors with HA-NLS-RARα and Myc-ISCA1 are constructed successfully. The interaction between ISCA1 and NLS-RARα is identified by co-immunoprecipitation and Western blotting in vitro.

关 键 词：核定位信号 受体 维甲酸 ISCA1 蛋白质相互作用 免疫共沉淀 

分 类 号：R733.71[医药卫生—肿瘤]