Construction and Expression of Enkaryotic Expression Vector of Canine Melanocortin Receptor-4

[Objective] The research aimed to construct the eukaryotic expression vector of canine melanocortin receptor-4 with myc and His tags and express in MDCK cells.[Method] With the genomic DNA of canine MC4R as template,the coding region of target gene was amplified by using PCR and T-A clone was made on the amplification products.After successful identification by enzyme digestion and sequencing,the target gene was subcloned into the eukaryotic expression vector pcDNA3.1-myc-His/A vector.The recombinant pcDNA3.1-myc-His/A-Canis MC4R was identified by enzyme digestion and sequencing.The recombinant was introduced into MDCK cells by using FuGENE HD transfecting technique.After 72 h culture,total RNA was extracted from cells and the expression of target gene was identified by using RT-PCR.Total cellular protein was extracted and the protein expression was detected by Western Blot.[Result] The recombinant eukaryotic expression vector pcDNA3.1-myc-His/A-Canis MC4R with tags was constructed.The similarity between the sequenced results and the reported sequence on GenBank website was 99%.The expression of target gene was detected by RT-PCR and Western Blot.[Conclusion] The eukaryotic expression vector pcDNA3.1-myc-His/A-Canis MC4R was successfully constructed and the recombinant could express in MDCK cell.