Construction of Canis Melanocortin Receptor-4 Eukaryotic Expression Vector and its Expression in COS-7 Cells

[Objective]To Construct the pcDNA3.1(+)-Canis MC4R expression plasmid and investigate its transient expression in COS-7 cells.[Methods]Using the canis MC4R DNA as template,designed the specific primers.The fragment of MC4R gene was amplified and then cloned into pMD18-T vector.The eukaryotic expression vector pcDNA3.1(+)-MC4R was constructed by ligating canis MC4R gene with pcDNA3.1(+) vector.The recombinant pcDNA3.1(+)-MC4R was digested by double restriction enzymes and sequenced.Then it was transfected into the COS-7 cells by FuGENE HD transfection reagent,from which total RNA was extracted.CDS of the canis MC4R was amplified by RT-PCR.[Results]The CDS of canis MC4R gene was cloned and it has a similarity of 99% with the reported sequence in GenBank.Through G418 filtration,We obtained the positive COS-7 cells.[Conclusion] The construction and the expression of eukaryotic plasmid pcDNA3.1(+)/MC4R have been achieved successfully.