rarα变异蛋白相互作用蛋白在哺乳细胞中的验证.doc

NLS-RARα相互作用蛋白在哺乳动物细胞中的验证 王翀， 王东生, 刘北忠, 郝坡，刘畅，金丹婷，钟梁，王春光 （重庆医科大学临床检验诊断学教育部重点实验室，重庆 400016） 摘要：目的 利用免疫共沉淀和蛋白质印记验证JTV1与NLS-RARα蛋白间的相互作用。方法 构建含HA标签的NLS-RARα融合蛋白的真核表达载体pCMV-HA-NLS-RARα, 及带有Myc标签的JTV1融合蛋白表达载体pCMV-Myc-JTV1酶切鉴定正确后，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 构建的真核表达载体鉴定正确，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单克隆抗体进行蛋白印记检测，可以检测到Myc-JTV1蛋白的表达。结论 成功构建了含HA-NLS-RARα、Myc-JTV1融合蛋白的真核表达载体，利用免疫共沉淀技术证实NLS-RARα与JTV1之间存在相互作用。 关键词：NLS-RARα；JTV1；蛋白质相互作用；免疫共沉淀 α by co-immunoprecipitation in mammalian cell WANG Chong, WANG Dong-sheng, LIU beizhong, etal. Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Faculty of Laboratory Medicine in Chongqing Medical University, (Chongqing, China 400016) Abstract Objective To explore the interaction between JTV1 and NLS-RARα by co-immunoprecipitation and western blotting. Methods Constructed the HA-tagged fusion protein (HA-NLS-RARα) eukaryotic expression vector and the Myc-tagged fusion protein (Myc-JTV1) eukaryotic expression vector, identified and then transfected into human embryo kidney 293 cells. Co-immunoprecipitation and western blotting was used to investigate the interaction between JTV1 and NLS-RARα. Results After being verified, eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, Myc-JTV1 protein was detected by western blotting with anti-Myc monoclonal antibody from the immunoprecipitared complex. Conclusion Eukaryotic expression vectors were constructed successfully. The interaction between JTV1 and NLS-RARα was identified by co-immunoprecipitation. Key Words: NLS-RARα, JTV1, Protein-protein interaction, Co-immunoprecipitation PML-RARα融合基因在急性早幼粒细胞白血病(APL)的发生发展中有重要作用，同时其作用并不始终以整体形式来发挥。NLS-RARα蛋白为PML-RARα融合蛋白在早期细胞内被中性粒细胞弹性蛋白酶（Neutrophil Elastase，NEα信号传导，具体的作用机制不明，为寻找与之相互作用蛋白我们运用酵母双杂交系统从白血病细胞cDNA文库中进行筛选，首次筛选得到JTV1蛋白与其有相互作用[2]，该蛋白在MAPK通路中有重要作用，同时作为氨基酰-tRNA合成酶复合体中3个非酶类蛋白之一发挥许多重要的生理作用。免疫共沉淀和蛋白质印记