肠出血性大肠杆菌Ⅲ型分泌系统ATP水解酶escN基因突变株的构建  

Construction of Type Ⅲ Secretion ATPase escN Gene Defective Mutant of Enterohemorrhagic Escherichia coli

作　　者：陈芳红[1,2] 李涛[2] 王慧[1,2] 

机构地区：[1]安徽医科大学,安徽合肥2300322 [2]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2015年第3期346-348,共3页Letters in Biotechnology

基　　金：国家重点基础研究发展计划(2015CB554202);国家自然科学基金(81401643)

摘　　要：目的:利用λ噬菌体的Red重组系统敲除肠出血性大肠杆菌O157∶H7的Ⅲ型分泌系统ATP水解酶Esc N,构建大肠杆菌esc N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,分别酶切连接于p UC19-kan质粒上,PCR获得中间嵌合卡那霉素抗性基因(带有FRT位点)的同源线性片段,利用质粒p KD46和p CP20介导的重组技术敲除esc N基因,并去除抗性标记;PCR及测序验证目的基因缺失后,测定缺失株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的esc N基因,突变株与野生株的生长曲线相近。结论:构建了Ⅲ型分泌系统缺陷菌株,为进一步研究Ⅲ型分泌系统因子在肠出血性大肠杆菌致病过程中的作用奠定了基础。Objective： To construct typeⅢ secretion ATPase esc N gene defective mutant of enterohemorrhagic E.coli（EHEC） O157∶H7 with λRed recombination system.Methods： The homologous regions which were clonedfrom EHEC O157∶H7 by PCR and kanamycin gene with two FRT sites were inserted into plasmid p UC19 to con-struct a recombinant vector.With the work of λRed system,target gene esc N was knockout.esc N gene deletion mu-tant was obtained and identified by PCR method and sequencing.Results： The typeⅢ secretion ATPase esc N de-fective mutant of EHEC was successfully constructed,and the growth curve of the mutant has no difference withwild strain.Conclusion： By constructing the esc N gene defective mutant,the experiments provide reference for thestudy of the secretion and translocation mechanism of typeⅢ secretion proteins in the future.

关 键 词：肠出血性大肠杆菌O157∶H7 Ⅲ型分泌系统 RED重组系统 

分 类 号：Q78[生物学—分子生物学]