The Interaction Between Decidual Protein Induced By Progesterone Depp And Sedlin Related To Spondyloepiphyseal Dysplasia Tarda

Objective To investigate the interaction region of Depp for Sedlin by yeast two-hybrid assay; To express fusion protein GST-Sedlin,GST pull-down assay was performed to detect the interaction between Depp and Sedlin in vitro;To construct the eukaryocyte expression vectors of Depp and Sedlin and COS7 cells were cotransfected to demonstrate whether Depp and Sedlin were colocalized in mammalian cells.Methods PCR method was used to amplify the N terminal 300bp and C terminal 339bp DNA fragments of Depp from plasmid pACT2-Depp,then the amplified fragments were inserted into pACT2 vector respectively to construct lack mutants pACT2-Depp-N300 and pACT2-Depp-C339.Each mutant was cotransformed into Y190 yeast competent cells with pAS-SEDL,then the yeast cells were cultured on SD (﹣Trp/﹣Leu/﹣His/+3-AT) plate, Clony-lift Filter Assay was performed to detect the activity ofβ-galactosidase, The blue clones were positive, which show protein-protein interactation.The whole sequence of Sedlin was amplified from pAS-SEDL by PCR method, the PCR product was inserted into the GST-tagged vector pGEX-3X to construct expression vector pGEX-3X-SEDL after digested with BamH I and EcoR I. E.coli BL21 cells were transformed with pGEX-3X-SEDL plamid to express fusion protein GST-Sedlin.Then GST pull-down assay was performed to detect the interaction between Depp and Sedlin in vitro.FLAG-Sedlin coding sequence was amplified and inserted into pCDNA3.1 to construct eukaryocyte expression vectors pCDNA-FLAG-SEDL. After confirmed by Restriction enzymatic analysis and DNA sequencing, COS7 cells were cotransfected with pCDGFP-Depp and pCDNA-FLAG-SEDL to demonstrate the colocalization of Depp and Sedlin by immunofluorescence.Results Each recombined plasmid was constructed correctly confirmed by restriction enzymatic analysis and DNA sequencing. Clony-lift Filter Assay showed that the yeast cells cotransformed with pACT2-Depp-C339 and pAS-SEDL became blue, were positive clones. GST pull-down assay indicated that Depp protein could interact with Sedlin protein in vitro. Depp and Sedlin were found to be colocalized at a concentrated region around nucleus in COS7 cells by immunofluorescence.Conclusion The critical region of Depp for its interaction with Sedlin located in C-terminal 112 amino acid residues; GST pull-down assay confirmed that Depp could interact with Sedlin in vitro; Immunofluorescence assay showed that protein Depp have a colocalization with Sedlin at a concentrated region around nucleus in COS7 cells,which indicated the interaction between Depp and Sedlin in mammalian cells.