Abstract:

BACKGROUND: Effects of melanocortin receptor (MCR) on in vivo energy balance arose more and more attention. MC3R gene mutation plays an important role in weight gaining, but there are few studies concerning MC3R, and no monoclonal antibody has been reported to detect MC3R expression at protein level. 
OBJECTIVE: To construct an expression vector pGEX-4T-1-cMC3R and to induce its expression in E. coli.
METHODS: Using the canis MC3R DNA as template, specific primers were designed. After the PCR amplification, the product was cloned into pMD18-T vector, and identified by double enzyme digestion. The target gene was subcloned into pGEX-4T-1 vector. The recombinant pGEX-4T-1-cMC3R was transferred to E. coli DH5α, and then it was identified with double restriction enzymes digestion analysis and DNA sequencing. The recombinant pGEX-4T-1-cMC3R which was identical to what released in GeneBank was transformed into E. coli BL21. The expression of canis MC3R protein was analyzed by Western blot after IPTG induction.
RESULTS AND CONCLUSION: The prokaryotic plasmid pGEX-4T-1-cMC3R was constructed successfully. Canis MC3R sequence was mainly identical to what released in GeneBank. After induction, the recombinant pGEX-4T-1-cMC3R could express canis MC3R fusion protein in E. coli BL21. This experiment not only provides material basis for construction of canis MC3R monoclonal antibody, but also benefits the structures and physiologic functions studies of canis MC3R protein.