The Preliminary Discussion On The Interactionof E2F1 Protein And Sedlin Protein

Objective:The interaction between E2F1 protein and Sedlin protein in vitro and in vivo was conducted. The results would provide a new kind of insight for targeted therapy of spondyloepiphyseal dysplasia tarda(SEDT).Methods:The full-length sequence of E2F1 was amplified with conventional polymerase chain reaction(PCR).The eukaryotic expression vector pc DNA3.1-E2F1-FLAG was builded. The correct recombinant plasmid was sent to sequencing after restriction enzyme digestion. pc DNA3.1-E2F1-FLAG was transient transfected into HEK 293 T cells. At the same time GST-Sedlin fusion protein was prepared. The interaction between E2F1 protein and Sedlin protein in vitro was detected by GST Pull-down technology.In COS-7 cells,pc DNA3.1-E2F1-FLAG and p CDGFP-Sedlin were transient transfected respectively.The localization of two proteins in the cells was observed.Then the above two plasmids were transient transfected together. The co-localization of two proteins in the cells was observed. pc DNA3.1-E2F1-FLAG and p CDGFP-Sedlin were transient transfected into HEK 293 T cells. The interaction between E2F1 protein and Sedlin protein in the mammalian cells was studied using co-immunoprecipitation assay.Results:The results of enzyme digestion analysis and sequencing showed the plasmid pc DNA3.1-E2F1-FLAG was constructed successfully.GST Pull-down experiment showed the interaction between E2F1 protein and Sedlin protein in vitro was exist. Observing under the fluorescence microscope, E2F1 protein in COS-7 cells was mainly distributed in the nucleus and not distributed obvious in the cytoplasm, However, Theexpression of Sedlin protein was obsevered both in the nucleus and in the cytoplasm. The co-localization of E2F1 and Sedlin in the nucleus by co-localization experiment. Co-immunoprecipitation assay showed the interaction between E2F1 protein and Sedlin protein in HEK 293 T cells was exists.Conclusion:In vitro, the interaction between E2F1 protein and Sedlin protein was exist. In COS-7 cells, the co-localization of E2F1 and Sedlin in the nucleus. In HEK 293 T cells,the interaction between E2F1 protein and Sedlin protein was exists.