Construction and Expression of Prokaryotic Expression Vector for Canine MC4R Gene

Objective To construct a prokaryotic expression vector for canine MC4R(cMC4R) gene and express recombinant MC4R protein.Methods MC4R gene was amplified by PCR using recombinant plasmid pcDNA3.1-myc-his / A-cMC4R as a template and cloned into prokaryotic expression vector pET-30a(+).The constructed recombinant plasmid pET-30a(+)-MC4R was transformed to E.coli Transetta(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Restriction analysis proved that recombinant plasmid pET-30a(+)-MC4R was constructed correctly.DNA sequencing proved that the homology of inserted sequence to that reported in GenBank was 99%,of which only the base T at site 777 of encoding region changed into C.The relative molecular mass of expressed protein was about 37 000.The expression level reached the maximum after induction for 4 h,which accounted for 8% of total somatic protein.The recombinant protein showed specific binding to anti-His monoclonal antibody.Conclusion The prokaryotic expression vector for cMC4R gene was successfully constructed,and recombinant cMC4R protein was expressed,which provided an antigen for preparation of polyclonal and monoclonal antibodies against cMC4R protein.