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-immuno precipiration in mammalian cells
王东生 1王春光 2王翀 2曹炬 2张国元 3王勇 3钟梁 2刘北忠2
作者信息
- 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;川北医学院附属医院检验科,四川,南充,637000
- 2. 重庆医科大学临床检验诊断学教育部重点实验室,重庆,400016
- 3. 川北医学院附属医院检验科,四川,南充,637000
- 折叠
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(NLSRARα)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-NIS-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-ILARα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NISRARd与ISCA1之间存在相互作用.
Abstract
AIM: To verify the interaction between ISCA1 and NLS-RARα by co-immunoprecipitation and Western blotting. METHODS: The eukaryotic expression vector pCMV-HA-NLS-RARα and pCMV-Myc-ISCA1 were constructed, identified and then transfected into human embryo kidney 293 cells. Co-immu-noprecipitation and Western blotting were used to investigate the interaction between ISCA1 and NLS-RARα. RESULTS: After being verified, eukaryotic expression rectors were co-transfected into HEK 293 cells, HA-NLS-RARα protein was then immunoprecipi-tated by anti-HA polyclonal antibody, and Myc-ISCA1 protein was detected by western blotting with anti-Myc monoclonal antibody from the immunoprecipitared complex. CONCLUSION: Eukaryotic 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/雏甲酸/ISCA1/蛋白质相互作用/免疫共沉淀Key words
nuclear localization signal/receptors/retinoic acid/ISCA1/protein-protein interaction/co-umnunopre-cipitation引用本文复制引用
基金项目
国家自然科学基金(30300449)
国家中医药管理局项目(02-03ZP52)
重庆医科大学基金(XBYB2007108)
重庆医科大学基金(XBYB2007104)
出版年
2009
NETL
NSTL
维普
万方数据