PML-C与GINS2蛋白相互作用的胞内验证

Evaluation of the Intracellular Interaction between GINS2 and PML-C inside Cells

目的 通过胞内实验验证PML-C与GINS2蛋白之间的相互作用.方法 将诱饵蛋白质粒pGBKT7-PML-C和文库蛋白质粒pACT2-GINS2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pCMV-HA-PML-C及pCMV-Myc-GINS2真核表达载体并共转染人胚肾293细胞,利用免疫共沉淀技术验证二者之间的相互作用.结果 pGBKT7-PML-C诱饵蛋白质粒和pACT2-GINS2靶蛋白质粒共转化AH109酵母菌后,可见蓝色阳性克隆生长;pCMV-HA-PML-C及pCMV-Myc-GINS2真核表达载体构建成功,共转染293细胞,抗HA多克隆抗体沉淀与HA-PML-C相互作用的蛋白复合物后,用抗Myc单克隆抗体进行Western印迹检测,可以检测到Myc-GINS2蛋白.结论 利用酵母双杂交和免疫共沉淀技术在胞内验证了PML-C与GINS2间存在相互作用.

Objective To evaluate the interaction between PML-C and GINS2 by Yeast two-hybrid and co-immunoprecipitation. Methods The plasmids of bait-protein and GINS2 protein were co-transformed into yeast AH109, to study the interaction of these proteins in live cells. Labeled fusion protein eukaryotic expression vectors were constructed and then co-transfected into human embryonic kidney 293 cells. Co-immunoprecipitation was used to evaluate intracellular interaction between PML-C and GINS2. Results Blue clones were formed in QDO/X-（x-gal plate. Eukaryotie expression vectors were constructed successfully and co-transfected efficiently into HEK 293 cells. Pulldown assay proved that HA-PML-C protein was immunoprecipitated by anti-HA polyclonal antibody, and Myc-GINS2 protein was measured by Western blotting with anti-Myc monoclonal antibody from the immunoprecipitated complex. Conclusion The intracellular interaction between PML-C and GINS2 was determined by Yeast two-hybrid and co-immunoprecipitation.