Thepreliminary Study On The Interaction Betweenphb1 Andclic1

Objective To investigate the interaction between phb1 and CLIC1 by yeast two-hybrid assay; To construct the eukaryocyte expression vectors with FLAG tagged phb1 and GFP tagged CLIC1;COS7 cells were contransfected to observed the colocalization of the specific situation; To contransfected to HEK293T cells for coimmuniprecipitation assay, Western blot was performed to investigate whether they are interacted or not.Methods The full length cDNA fragment of phb1 in pLenti6-phb1 vector was used as template to be amplified with PCR , PCR product and pGADT7vector were digested by appropriate restriction enzymes separatly, and ligated to construct the yeast expression vector pGADT7-phb1;pGADT7-phb1and pGBKT7 vector were digested by restriction enzymes , and ligated to construct the yeast expression vector pGBKT7-phb1;PCR method was used to amplify the full length cDNA fragment of CLIC1 gene . PCR product and pGBKT7 vector were digested by BamHⅠ, and ligated to construct the yeast expression vector pGBKT7-CLIC1; pGADT7-CLIC1 was constructed with the same method. The correct sequencing of yeast expression vector groups were cotransformed to the yeast AH109,then the yeast cells were cultured on SD/-Leu/-Trp/-His/+3-AT/X-α-gal plates selected the positive clones of bule screen , which were shown the two proteins may be interact,Clony-lift Filter Assay was further performed to detect the activity ofβ-galactosidase.which confirmed protein-protein interaction. Phb1 gene was cloned into pCDNA3 with FLAG-tagged , this eukaryocyte expression vector was transfected into the mammalian cell lines ,then observed the localization of the product of phb1 gene in fluorescent microscopy; COS7 cells were cotransfected with pCDNA3-FLAG-phb1 and pCDGFP-CLIC1 to demonstrate the colocalization of phb1 and CLIC1 by immunofluorescene; HEK293T cells were cotransfected with pCDNA3-FLAG-phb1 and pCDGFP-CLIC1 and extractec cells lysis, we try to investigate whether phb1 and CLIC1 can interact each other in mammalian cells with coimmuniprecipitation assay .Results Each recombined plasmid was constructed correctly confirmed by restriction enzymatic analysis and DNA sequencing. Nutrition screening andα, -galactosidase activity tests showed that there were positive clones, it was that phb1 and CLIC1 can interact with each other in the yeast AH109. pCDNA3-FLAG-phb1 and pCDGFP-CLIC1were cotransfected into COS7 cells, phb1 and CLIC1were fonud to be colocalized at a cncentrated region around nucleus in COS7 cells by immunofluorescene. It was demonstrated that phb1 and CLIC1 interaced with each other in HEK293T cells by coimmuniprecipitation assay and West blot .Conclusion Used yeast-two-hybrid system 3 to preliminary understand the interactation between phb1 and CLIC1. Immunofluorescene assay observed that phb1have a colocalization with CLIC1 at a concentrated region around nucleus in COS7 cells. Coimmuniprecipitation assay further confimed that phb1 and CLIC1 interacted with each other in HEK293T cells.