肠出血性大肠杆菌O157：H7 z1445基因缺失株的构建

Construction of z1445 Gene Mutant Strain of Enterhemorrhagic E.coli O157∶H7

目的:利用Red重组系统敲除肠出血性大肠杆菌(EHEC)O157∶H7的z1445基因,构建大肠杆菌z1445基因缺失突变株。方法:以O157∶H7为模板,PCR扩增目的基因两侧的同源臂序列,分别经酶切后连接到p UC19-kan质粒的卡那霉素抗性基因kan两侧,PCR获得中间嵌合kan基因(带有FRT位点)的同源线性片段,利用质粒p KD46敲除z1445基因,利用质粒p CP20去除抗性标记基因;PCR鉴定及测序验证目的基因缺失后,测定缺失株及野生株的生长曲线。结果:敲除了z1445基因,突变株与野生株的生长曲线接近。结论:构建了z1445基因缺陷型菌株,为进一步分析z1445基因在O157∶H7与宿主的相互作用中发挥的作用提供了材料。

Objective： To construct z1445 gene defective mutant of enterhemorrhagic E.coli（EHEC） O157：H7 with bred recombination system. Methods： The homologous regions which were cloned from EHEC O157：H7 by PCR and kanamycin gene with two FRT sites were inserted into pUC19 to construct a recombinant vector. With the work of bRed system, target gene z1445 was knockout, z1445 gene deletion mutant was obtained and identi- fied by PCR method and sequencing. Results： z1445 gene deletion mutant of EHEC was successfully constructed, and the growth curve of the mutant has no difference with wild strain. Conclusion： By constructing the z1445 gene defective mutant, the experiments provide reference for the study of the function of z1445 gene.