| 摘要: |
| 目的 通过胞内外实验验证谷氨酸氨连接酶(GLUL)与带有核定位信号的维甲酸受体α(NLS-RARα)之间的相互作用。方法 将表达GLUL靶蛋白和NLS-RARα诱饵蛋白的两种重组表达质粒共同转化入AH109酵母菌,采用一对一的酵母双杂交技术验证它们在活细胞内的相互作用；通过构建GLUL及NLS-RARα蛋白标签融合表达载体,共转染至人胚肾HEK 293细胞,利用免疫共沉淀技术在细胞外验证它们之间的相互作用。 结果 GLUL靶蛋白和NLS-RARα诱饵蛋白质粒共转化AH109酵母菌后,可见蓝色的阳性克隆；GLUL蛋白及NLS-RARα标签融合表达载体构建成功,共同转染至HEK 293细胞,采用抗HA多克隆抗体免疫沉淀HA-NLS-RARα相互作用蛋白复合物后,抗c-Myc单克隆抗体免疫印迹检测,检测到GLUL-cMyc蛋白。结论 采用酵母双杂交和免疫共沉淀技术成功地在胞内外验证了GLUL与NLS-RARα之间存在特异性的相互作用。 |
| 关键词: 维甲酸受体α 核定位信号； 谷氨酸氨连接酶 蛋白质相互作用 双杂交系统技术 免疫共沉淀 |
| DOI：10.3724/SP.J.1008.2010.0468 |
| 投稿时间：2009-11-30修订日期：2010-04-19 |
| 基金项目: 国家自然科学基金(30300449)，国家中医药管理局面上项目(02-03ZP52)，重庆医科大学课题(XBYB2007104). |
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| Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells |
| WU Yan, LIU Bei-zhong*, WANG Chong, ZHONG Liang, ZHU Dan, WANG Chun-guang, JIN Dan-ting |
| (Key Laboratory of Clinical Medical Diagnostics, Ministry of Education, Faculty of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China) |
| Abstract: |
| Objective To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method. Methods The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells. Co-immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro. Results Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody. Conclusion The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation. |
| Key words: retinoic acid receptor α nuclear localization signals glutamate-ammonia ligase protein interaction two-hybrid system techniques co-immunoprecipitation |
