带核定位信号的RARα与谷氨酸氨连接酶蛋白相互作用的胞内外验证

Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells

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HEK293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HEK293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。

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.