PML coiled-coil结构域与RAN结合蛋白9相互作用的验证

Identification of Protein-Protein Interaction between Coiled-Coil Region of Promyelocytic Leukemia and RAN Binding Protein 9

目的验证前髓细胞性白血病的卷曲螺旋结构域（PML—C）和RAN结合蛋白9（RANBP9）之间的相互作用。方法构建分别表达诱饵蛋白PML—C和靶蛋白RANBP9的载体pGBKT7-PML-C和pACT2-RANBP9，然后转人酵母AH109，培养3～5d后对其是否有胞内相互作用进行检测。将目的片断PML-C和RANBP9再次构建于真核生物表达载体pCMV—HA和pCMV—myc里，然后共转染人胚肾293细胞里（HEK293），最后对其是否有体外相互作用通过免疫共沉淀和免疫印迹进行分析。结果在共转化了质粒pGBKT7-PML—C和pACT2-RANBP9的AH109酵母平板里观察到蓝色菌落生长。用抗HA多克隆抗体对共转染过重组质粒的HEK293细胞的蛋白提取物进行免疫共沉淀，再用抗myc单克隆抗体作为一抗进行免疫印迹，最终检测出融合蛋白myc—RANBP9条带。结论酵母双杂交实验验证了PML—C和RANBP9之间存在胞内相互作用，同时免疫共沉淀实验也从体外验证了它们之间的相互作用。

Objective To demonstrate the interaction between coiled-coil region of promyelo- cytic leukemia （PML-C） and RAN binding protein 9 （RANBP9） . Methods Plasmids pGBKT7- PML-C and pACT2-RANBP9 that express bait protein PML-C and target protein RANBP9 respectively were constructed and co-transformed into yeast AH109 afterwards. The interaction of them in yeast AH109 was detected by yeast two hybrid assay after incubation from 3 to 5 days onwards. Fragments PML-C and RANBP9 were constructed into eukaryotic expression vector pCMV-HA and pCMV-myc separately and the recombinants were co-transfected into HEK293 cells. The interac- tion of them in HEK293 was detected through co-immunoprecipitation and western blot analy- sis. Results Blue colonies were observed on AH109 plate which were co-transformed with Plasmids pGBKTT-PML-C and pACT2-RANBP9. Fusion protein myc-RANBP9 was detected by western blot with anti-Myc monoclonal antibody after co-immunoprecipitaion with anti-HA polyclonal antibod- y. Conclusion The intracellular interaction between PML-C and RANBP9 was identified by yeast two-hybrid system and the interaction of them in vitro was identified by co-immunoprecipitation furthermore.