The Preliminary Study Of The Interaction Between Phb2 And Clic1

Objective To investigate the interaction between phb2 and CLIC1 by yeast two-hybrid assay; To construct the FLAG tag eukaryocyte expression vector for human wild phb2 and GFP tag eukaryocyte expression vector for human CLIC1 ;COS7 cells were cotransfected to observe the colocalization of the specific situation; To be cotransfected to HEK293T cells for coimmuniprecipitation assay, Western blot was performed to investigate whether they interacted or not.Methods The full length cDNA fragment of phb2 in pLenti6-phb2 vector was used as template to be amplified with PCR , PCR product and pGADT7vector were digested by appropriate restriction enzymes separately, and ligated to construct the yeast expression vector pGADT7-phb2;in the same way ,we constructed pGBKT7 -phb2;PCR was used to amplify the full length cDNA fragment of CLIC1 gene . PCR product and pGBKT7 vector were digested by appropriate restriction enzymes separately, and ligated to construct the yeast expression vector pGBKT7- CLIC1; pGADT7- CLIC1 was constructed with the same method. The correct sequencing of yeast expression vectors were cotransformed to the yeast AH109,and then the yeast cells were cultured on SD/-Leu/-Trp/-His/+3-AT/X-α-gal plates to select the positive bule clones , which showed the two proteins interacted.Clony-lift Filter Assay was further performed to detect the activity ofβ-galactosidase,which showed protein-protein interaction.Phb2 gene with FLAG-tag was cloned into pCDNA3 , and this eukaryocyte expression vector was transfected into the mammalian cell lines to observe the localization of the product of phb2 gene by virtue of fluorescent microscopy; COS7 cells were cotransfected with pCDNA3-FLAG-phb2 and pCDGFP- CLIC1 to demonstrate the colocalization of phb2 and CLIC1 by immunofluorescence; HEK293T cells were cotransfected with pCDNA3-FLAG-phb2 and pCDGFP- CLIC1 and extracted cell extracts to investigate whether phb2 and CLIC1 can interact with each other in mammalian cells with coimmuniprecipitation assay .Results Each Constructed plasmid was correctly confirmed by restriction enzymatic analysis and DNA sequencing. Nutrition screening ,α-galactosidase activity tests andβ-galactosidase activity tests showed positive clones that phb2 and CLIC1 interacted with each other in the yeast AH109. pCDNA3-FLAG-phb2 and pCDGFP- CLIC1 were cotransfected into COS7 cells, which were fonud to be colocalized in the cytoplasm of COS7 cells with patchy distribution by immunofluorescence. It was demonstrated that phb2 and CLIC1 interacted with each other in HEK293T cells by coimmuniprecipitation assay and Western blot .Conclusion the results of yeast-two-hybrid assay ware to preliminary confirm the interaction between phb2 and CLIC1. by virtue of Immunofluorescence assay, We observed that phb2 had a colocalization with CLIC1 with patchy distribution in the cytoplasm of COS7 cells . Coimmuniprecipitation assay further confirmed that phb2 and CLIC1 interacted with each other in HEK293T cells.