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目的通过胞内外试验验证含Coiled-coil结构的PML结构域(PML-C)与CNPY2蛋白的相互作用。方法将质粒pACT2-CNPY2和pGBKT7-PML-C共转化入酵母菌AH109,通过酵母双杂交技术检测CNPY2蛋白和PML-C在AH109细胞内的相互作用;构建重组质粒pCMV-HA-PML-C和pCMV-Myc-CNPY2,共转染HEK293细胞,采用Western blot法检测细胞中CNPY2蛋白的表达;免疫共沉淀技术从胞外验证两种蛋白的相互作用。结果 pACT2-CNPY2和pGBKT7-PML-C质粒共转化入AH109酵母菌后,可见蓝色阳性克隆;重组质粒pCMV-HA-PML-C和pCMV-Myc-CNPY2共转染的HEK293细胞的全细胞裂解液可与鼠抗c-MYC单抗特异性结合;经免疫共沉淀可检测到与PML-C相互作用的蛋白复合体。结论通过酵母双杂交试验和免疫共沉淀技术证实,PML-C与CNPY2蛋白存在相互作用。
Objective To verify the interaction between PML-C with Coiled-coil structure and CNPY2 protein in vitro and in vivo. Methods The pACT2-CNPY2 and pGBKT7-PML-C plasmids were co-transformed into yeast AH109. The yeast two-hybrid assay was used to detect the interaction between CNPY2 protein and PML-C in AH109 cells. The recombinant plasmid pCMV-HA-PML- pCMV-Myc-CNPY2 were co-transfected into HEK293 cells and the expression of CNPY2 protein was detected by Western blot. The co-immunoprecipitation assay was used to verify the interaction between the two proteins. RESULTS: Positive clones were observed after co-transformation of pACT2-CNPY2 and pGBKT7-PML-C plasmids into AH109 yeast. Whole cell lysis of HEK293 cells co-transfected with pCMV-HA-PML-C and pCMV-Myc-CNPY2 The liquid can specifically bind to mouse anti-c-MYC monoclonal antibody; the protein complex that interacts with PML-C can be detected by co-immunoprecipitation. Conclusion The yeast two-hybrid assay and co-immunoprecipitation demonstrate that PML-C interacts with CNPY2 protein.