Construction of a Prokaryotic Expression Plasmid Encoding the mtrC Gene of N. gonorrhoeae and Its Expression in E. coli / 中华皮肤科杂志

ABSTRACT

Objective To construct a prokaryotic expression plasmid pET-28a(+) encoding the multiple transferable resistance C (mtrC) gene of N. gonorrhoeae and express it in E.coli DE3, in order to provide a model to study the pathogen's resistance mechanisms to antimicrobial hydrophobic agents. Methods The mtrC gene of N. gonorrhoeae was amplified by polymerase chain reaction from reference strains,cleaved with restriction endonuclease, and then cloned into the prokaryotic expression plasmid pET-28a (+) to construct the recombinant pET-mtrC. This was confirmed by cleavage of restriction endonuclease and DNA sequencing. The recombinant pET-mtrC was transformed into E.coli DE3 to express the protein MtrC with induction by IPTG. Results The mtrC gene in the recombinant pET-mtrC showed 99.5% homology with the reference sequence in GeneBank (U14993). A 48.5 kD fusion protein was identified by SDS-PAGE. Conclusions The successful construction of a prokaryotic plasmid encoding the mtrC gene of N. gonorrhoeae and its expression in E.coli may facilitate the development of a monoclonal antibody to the MtrC protein and help to investigate the mechanism of the mtr efflux system of N. gonorrhoeae.