The Interaction Between COX6B1and Sedlin And A Pilot Study On The Influence Of COX Caused By COX6B1

Objective Investigate the interaction of COX6B1for Sedlin by yeast two-hybridassay; Construction the eukaryocyte expression vectors of COX6B1protein and twopoint mutants R20H、D74A with FLAG tags, transfected into COS7cells to observethem single localization and difference with the wild type. Construction theeukaryocyte expression vectors of Sedlin protein with GFP tags, transfected intoCOS7cells to observe its localization,co-transfected with COX6B1protein and twopoint mutants R20H、D74A with FLAG tags into COS7cells together to observe theco-localization of them in mammals cells,respectively; co-transfected COX6B1protein tagged with FLAG and GPF-tagged Sedlin protein into HEK293T cellstogether for immunoprecipitation to detect whether COX6B1protein can interact withSedlin protein in mammalian cells.Methods The full length cDNA fragment of COX6B1from plasmid was used toamplify with PCR, then the fragment was inserted into pGBKT7vector to constructexpression vector pGBKT7-COX6B1, then construct expression vectors pGBKT7-COX6B1R20H、pGBKT7-COX6B1D74A from plasmid pGBKT7-COX6B1. Eachvectors was co-transformed into AH109yeast competent cells with pGADT7-SEDL,then the yeast cells were cultured on （﹣Trp/﹣Leu/﹣His/＋3-AT）/X-α-gal plate,The blue clones were X-α-gal activity positive clone.The whole sequence of COX6B1was amplified from plasmid pGBKT7-COX6B1by PCR method, with the upstream primer containing FLAG tags, then thefragment was inserted into pCDNA3.1vector to construct expression vectorpCDNA3.1-FLAG-COX6B1. Analogously, expression vectorspCDNA3.1-FLAG-COX6B1R20H、 pCDNA3.1-FLAG-COX6B1D74A were constructed from plasmids pGBKT7-COX6B1R20H、 pGBKT7-COX6B1D74A.They were transfected into COS7cells and observed their localization in cells withwild-type difference under a fluorescence microscope, then with pCDGFP-SEDLco-transfected into COS7cells to observe their co-localizations. PlasmidspCDNA3.1-FLAG-COX6B1and pCDGFP-SEDL were co-transfected into HEK293T cells, extracted cell lysatefor immunoprecipitation, then detected whetherCOX6B1protein can interact with Sedlin protein in mammalian cells by Western blot.Results Each recombined plasmid was constructed correctly confirmed byrestriction enzymatic analysis and DNA sequencing. Nutrition Screening andα-galactosidase activity tests indicated COX6B1wild-type and two point mutantsR20H、D74A protein can interact with Sedlin protein. The product of COX6B1genewas found to be mainly localized in the cytoplasm, not to be distributed in the nucleusobviously, the two point mutants R20H、D74A protein are similar to the wildtype.Sedlin protein was found to be localized in the cytoplasm and nucleus.Immunofluorescence microscopy experiments observed COX6B1protein and Sedlinprotein co-localized at a concentrated region around the nucleus, similar as the twopoint mutants. Immunoprecipitation and Western blot proved that Sedlin protein caninteract with COX6B1protein in HEK293T cells.Conclusion Yeast two-hybrid assay indicated that protein COX6B1and two pointmutants could interact with protein Sedlin. Immunofluorescence assay showed thatprotein COX6B1have a colocalization with Sedlin at a concentrated region aroundperinuclear in COS7cells, which indicated the interaction between COX6B1andSedlin in mammalian cells; the two point mutants R20H、D74A protein are similar tothe wildtype. Co-Immunoprecipitation experiments show that COX6B1protein caninteract with Sedlin protein in mammalian cells.