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带核定位信号的RARα与JTV1蛋白相互作用的验证实验 被引量：5

Identification of the Interactions Between JTV1 and NLS-RARα in vivo and in vitro
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摘要 目的通过实验验证带有核定位信号的维甲酸受体α(NLS-RARα)与JTV1蛋白之间的相互作用。方法将表达NLS-RARα诱饵蛋白和JTV1靶蛋白的两种重组表达质粒共转化AH109酵母菌,通过一对一的酵母双杂交技术验证两者在活细胞内的相互作用;构建NLS-RARα及JTV1蛋白标签融合表达载体并共转染人胚肾293细胞,利用免疫共沉淀技术在体外验证二者之间的相互作用。结果NLS-RARα诱饵蛋白和JTV1靶蛋白质粒共转化AH109酵母菌后,可见蓝色阳性克隆;NLS-RARα及JTV1蛋白标签融合表达载体构建成功,共转染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单克隆抗体进行免疫印迹检测,可以检测到Myc-JTV1蛋白。结论利用酵母双杂交和免疫共沉淀技术验证了NLS-RARα与JTV1间存在相互作用。 Objective To identify the interactions between JTV1 and NLS-RARα by Yeast two-hybrid and co- immunoprecipitation. Methods The plasmids of bait-protein and JTV1 protein were cotransformed into yeast AH109 to investigate their interactions in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and then cotransfected into human embryo kidney 293 cells. Co-immunoprecipitation was used to investigate the interactions between NLS-RARα and JTV1 in vitro. Results Blue clones were found in QDO/X-α- gal plates. Eukaryotic expression vectors were co-transfected into HEK 293 cells. The 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 The interactions between NLS-RARα and JTV1 are identified by Yeast two-hybrid and co-immunoprecipitation.
作者 王翀 王东生 刘北忠 郝坡 刘畅 金丹婷 钟梁 王春光
机构地区 重庆医科大学医学检验系临床检验诊断学教育部重点实验室
出处 《四川大学学报（医学版）》 CAS CSCD 北大核心 2009年第3期382-384,402,共4页 Journal of Sichuan University(Medical Sciences)
基金 国家自然科学基金(批准号30300449) 国家中医药管理局课题(02-03ZP52) 重庆医科大学课题(XBYB2007104 XBYB2007108)资助
关键词 NLS-RARα JTV1 酵母双杂交 免疫共沉淀 蛋白质相互作用 NLS-RARα JTV1 Yeast two-hybrid Co-immunoprecipitation Protein-protein interaction
分类号 Q78 [生物学—分子生物学]
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共引文献3

同被引文献45

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四川大学学报（医学版）
2009年 第3期

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