淋病奈瑟菌mtrC膜蛋白基因的克隆和表达

Construction of a Prokaryotic Expression Plasmid Encoding the mtrC Gene of N. gonorrhoeae and Its Expression in E. coli

目的构建淋病奈瑟菌膜蛋白mtrC表达质粒,探讨淋病奈瑟菌耐药的检测和耐药机制。方法从淋病奈瑟菌标准株中扩增淋病奈瑟菌膜蛋白mtrC基因片段。酶切后插入pET-28a(+),构建重组表达质粒pET-mtrC。通过质粒双酶切和DNA测序证实该重组质粒构建正确。重组质粒转化入大肠杆菌 DE3中。经IPTG诱导表达蛋白。结果经酶切鉴定和测序分析,质粒构建正确。核苷酸序列与GeneBank (U14993)公布的序列相比较,同源性达到99．5％。通过IPTG诱导,SDS—PAGE可检测到约48 500大小的融合蛋白,与预测分子量相同。结论淋病奈瑟菌膜蛋白mtrC原核表达质粒构建和表达成功,为研究其 mtrC外排系统的耐药机制打下基础。

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.