免疫共沉淀法测定14-3-3ε蛋白与Cdc25B的相互作用

Study on the interaction between 14-3-3ε protein and Cdc25B determined by co-precipitation

目的研究14-3-3ε蛋白与Cdc25B的相互作用及其作用位点,为小鼠卵母细胞减数分裂G2期阻滞的机制研究提供实验依据。方法本实验依次构建pcDNA3.1-ZEO-HA-14-3-3ε、pEGFP-Cdc25B-WT、pEGFP-Cdc25B-Ser321A、pEGFP-Cdc25B-Ser321D表达载体,分别转染HEK293细胞,分为四组,分别为Cdc25B-vector+14-3-3ε组(空载体组),Cdc25B-WT+14-3-3ε组,Cdc25B-Ser321A+14-3-3ε组,Cdc25B-Ser321D+14-3-3ε组。通过免疫共沉淀法获得各组细胞裂解液及免疫沉淀物,用Western blotting检测Cdc25B-vector、Cdc25B-WT、Cdc25B-Ser321A、Cdc25B-Ser321D与14-3-3ε蛋白是否有共沉淀,如有共沉淀,说明两蛋白存在相互作用。结果本实验构建的4种表达载体经验证均构建成功。免疫共沉淀实验得出14-3-3ε能与野生型Cdc25B结合,当Cdc25B的S321突变成S321A时,这种结合不发生;而具有模拟磷酸化作用的Cdc25B-S321D也能与14-3-3ε结合。结论Cdc25B S321是14-3-3ε蛋白与Cdc25B相互作用的特异性结合位点,此结合位点可能成为14-3-3ε蛋白与Cdc25B共同调控小鼠卵母细胞减数分裂进程及G2期阻滞的重要靶点,为后期哺乳动物生殖细胞的相关研究奠定实验基础。

Objective To investigate the interaction between 14-3-3εand Cdc25B and its sites and provide an experimental basis for the mechanism of mouse oocytes arresting in G2 phase in meiosis.Methods In this experiment,pcDNA3.1-ZEO-HA-14-3-3ε,pEGFP-Cdc25B-WT,pEGFP-Cdc25B-Ser321A and pEGFP-Cdc25B-Ser321D were constructed and transfected into HEK293 cells,respectively,which were named as Cdc25B-vector+14-3-3εgroup(empty vector group),Cdc25B-WT+14-3-3εgroup,Cdc25B-Ser321A+14-3-3εgroup,Cdc25B-Ser321D+14-3-3εgroup.The cell lysates and immunoprecipitates were obtained by co-immunoprecipitation.The co-precipitation of Cdc25B-vector,Cdc25B-WT,Cdc25B-Ser321A,Cdc25B-Ser321D with 14-3-3εwere determined by Western blotting.If there is co-precipitation,there is the interaction between the two proteins.Results The four expression vectors were successfully constructed in this experiment.The co-immunoprecipitation experiment showed that 14-3-3εwas binded to wild-type Cdc25B.When S321 was mutated into S321A in Cdc25B,the binding was not found.Cdc25B-S321D with mimetic phosphorylation was also binded to the 14-3-3ε.Conclusion Cdc25B S321 is a specific binding site for the interaction of 14-3-3εprotein and Cdc25B.The 14-3-3εprotein and Cdc25B may co-regulate mouse meiotic process and G2 phase arrest by Cdc25B S321,which lay the experimental foundation for the related research of mammalian germ cells.