c-Fos不同结构域的真核表达及功能鉴定

目的：克隆c-Fos蛋白不同结构域基因片段，构建其含FLAG标签的真核表达载体，并鉴定其功能。方法：用PCR方法扩增获得c-Fos不同结构域片段基因，定向克隆至本实验室保存的FLAG-peDNA3．0载体中，瞬时转染293T细胞进行表达，并以本实验室保存的HA-e-Jun与FLAG-c-Fos不同结构域片段免疫共沉淀鉴定其相互作用区域，以佐证所构建的c-Fos结构域克隆的正确性。结果：测序结果表明c-Fos不同结构域序列正确，Western印迹显示可以在真核细胞中获得表达，并且免疫共沉淀结果显示c-Jun可以与c-Fos的bZIP区域特异性结合。结论：构建并表达了c-F0s不同结构域片段，为进一步研究与c-Fos相互作用的蛋白及其区域定位奠定了基础。

Eukaryotic Expression and Functional Characterization of Different Domains of c-Fos

Objective： To clone different domains of c-Fos in an eukaryotic vector harboring FLAG tag and to characterize their functions. Methods： Three different domains DNA were amplified by PCR method, and then were inserted into the eukaryotic expression vector FLAG-pcDNA3.0. The recombinant expression vectors were tran- siently transfected into 293T cell lines. The interaction between HA-tagged c-Jun and different FLAG-tagged e-Fos domains were demonstrated by co-immunoprecipitation. Results： The sequences of the cloned FLAG-tagged c-Fos domains were confirmed by DNA sequencing. The expression of the e-Fos domains was detected out by Western blot after collecting the cell lysate. Co-immunopreeipitation assays showed that c-Jun could interact specif- ically with the bZIP domain of c-Fos. Conclusion： Different domains of c-Fos were successfully constructed, which laid the foundation for further investigating the proteins that interact with the c-Fos.