Construction and Expression of Canis MC4R-D90N Mutation in Eukaryotic Cells

[Objective] The research aimed to construct the expressional vector with myc and His tags of canis MC4R-D90N gene and investigate its expression in MDCK cells.[Method] Using Canis genome DNA as template,the CDS fragment of MC4R-D90N was amplified by overlap PCR gene and then cloned into pMD18-T vector.After correct identification,the target gene was subcloned into pcDNA3.1-myc-His/A vector.The recombinant pcDNA3.1-myc-His/A-Canis MC4R-D90N was digested by double restriction enzymes and sequenced.Then it was transfected into the MDCK cells by FuGENE HD transfection reagent.After 72.0 hours culture,the expression of Canis MC4R-D90N gene was analyzed by RT-PCR and Western blot.[Result] The recombinant pcDNA3.1-myc-His/A-Canis MC4R-D90N mutation was achieved.MDCK cells that brought recombinant plasmid of D90N mutation were obtained by transfection reagent.The target sequence of Canis MC4R-D90N was amplified by RT-PCR and the expression of fusion protein was detected.[Conclusion] The construction and the expression of eukaryotic plasmid pcDNA3.1-myc-His/A-cMC4R-D90N were achieved successfully.And the recombination could be expressed in MDCK cells.