大肠杆菌O157∶H7 EDL933wz3672基因缺失突变体的构建

Construction of z3672 deletion mutant of EHEC O157∶H7 EDL933w

目的:构建EHEC O157∶H7 EDL933wz3672基因缺失突变体。方法:根据已知的O157∶H7 EDL933基因组全序列,采用pKOBEG介导的Red重组系统,筛选卡那霉素抗性基因替换z3672基因的阳性克隆,然后FLP位点特异性重组去除两端带有FRT位点的卡那霉素抗性基因,最后通过PCR和测序手段证实。结果:获得了z3672基因缺失而且不含有卡那霉素抗性基因的EHEC O157∶H7 EDL933w突变菌株。结论:为进一步研究O157∶H7的致病机制奠定了基础。

Objective：To construct a z3672 knockout mutant of EHEC O157：H7 EDL933w. Methods：Based on the whole genome sequence of O157：H7 EDL933, the z3672 gene was knocked out using the Red recombination system media- ted by pKOBEG. With the help of the Red recombinant system, z3672 gene of EHEC O157：H7 EDL933w was replaced by kanamycin resistant gene in vivo. Then kanamycin gene flanked by FRT （ FLP recognition target） sites was eliminated by site-specific recombination of FLP. Finally, the strain of z3672 deletion mutant was verified by PCR and sequencing. Re- suits：The z3672 knockout mutation strain EHEC O157：t/7 EDL933wAz3672 was constructed. Conclusion：The research re- sults will be of great value for the further research on pathogenesis of EHEC O157： H7.