The Plasmid Construction Of Metallothionein Genes And Analysis Of Overexpression In Cells

Objective To investigate the biological functions of MT genes, we constructedeukaryotic expression vectors, tagged with FLAG. The plasmid was transientlytransfected into mammalian cells to observe the localization of expressed protein. Theplasmid of MT2A was transiently cotransfected with CLIC1into mammalian cells todefine whether two expressed proteins were colocalized. The plasmid of MT2A andCLIC1were transiently cotransfected into mammalian cells to performco-immunoprecipitation assay. To express fusion protein GST-MT2A,GST pull-downassay was performed to detect the interaction between MT2A and CLIC1in vitro.Methods PCR method was used to amplify the full-length of MT. PCR product ofMT cDNA and vector were separately digested by appropriate restriction enzymes, and then ligated into pGADT7-MT, pCDNA3.1-MT-His6, and pCDNA3.1-MT-FLAG. Allplasmids were confirmed by restriction enzyme analysis and DNA sequencing.Plasmid was transiently transfected into COS7cells to show the localization ofexpressed proteins. MT2A was cotransfected with CLIC1into COS7cells and imageswere obtained by fluorescence microscope to investigate whether MT2A protein wascolocalized with CLIC1. Finally, the interaction of MT2A with CLIC1were performedby co-immunoprecipitation assay. E.coli BL21cells were transformed withpGEX-3X-MT2A plasmid to express fusion protein GST-MT2A.Then GST pull-downassay was performed to detect the interaction between MT2A and CLIC1in vitro.Results Each recombined plasmid was constructed correctly confirmed by restrictionenzymatic analysis and DNA sequencing. MT2A was mainly localized in cytoplasm ofCOS7cells. MT2A and CLIC1were not found to be colocalized in COS7cells byimmunofluorescence. In co-immunoprecipitation assay,MT2A and CLIC1could notinteraction in HEK293T cells. GST pull-down assay indicated that MT2A protein couldnot interact with CLIC1protein in vitro.Conclusion In COS7cells, MT2A was not observed obvious colocalized with CLIC1by fluorescence microscope. Co-immunoprecipitation experiment suggested MT2A wasnot interacted with CLIC1in HEK293T cells. GST pull-down assay confirmed thatMT2A could not interact with CLIC1in vitro. Objective To investigate interaction of DG with Sedlin, we constructed eukaryoticexpression vectors, tagged with FLAG. The plasmid was transiently transfected intomammalian cells to observe the localization of expressed protein. The plasmid of DAG1and Sedlin were transiently cotransfected into mammalian cells to investigate whetherDG was colocalized with Sedlin.Methods Vectors with full-length DAG1was used as template for amplification.PCR product of DAG1cDNA and vector were separately digested by appropriaterestriction enzymes, and then ligated into pCDNA3.1-DAG1-FLAG. Plasmid wastransiently transfected into COS7cells to show the localization of expressed proteins.DAG1was cotransfected with Sedlin into COS7cells and images were obtained byfluorescence microscope to investigate whether DG was colocalized with Sedlin.Results We obtained plasmids in-framed with DAG1gene. The product of DAG1gene was found to be localized in the cytoplasm and nucleus, especially distributed inthe membrane obviously. After the Sedlin recombined vector was cotransfected intoCOS7cells, the distribution of DG protein in the cells appeared to concentrate nearnucleus.Conclusion The product of DAG1gene was found to be localized in the cytoplasmand nucleus, especially distributed in the membrane obviously. After the Sedlinrecombined vector was cotransfected into COS7cells, the distribution of DG protein inthe cells appeared to concentrate near nucleus.