EHEC O_(157)∶H_7肠粘附力增强突变株的构建

Construction of mutant strain with increased adherence to caco-2 cells in enterohemorrhagic Escherichia coli O_(157):H_7

〔目的〕构建对肠上皮细胞粘附力增强的肠出血性大肠埃希菌O157∶H7(EHECO157)突变株 ,为体内、体外研究其粘附及其粘附机制提供可行的手段。〔方法〕分别将pSC10 1和mini -Tn5Km2通过电穿孔转化和结合导入O157∶H7Sakai菌株中 ,筛选粘附力增强的转化子感染Caco -2细胞株和实验小鼠 ,观察Caco -2单层细胞中的微菌落数量和小鼠粪便中突变株脱落的时程。〔结果〕经诱变 ,共分离筛选出 8株突变株 ,其形成微菌落的数量大于野毒株至少 3倍以上 ;8个突变株中有 6株转座子的插入位置为E .coliK -12同源区的yhiE基因处 ,其突变株在小鼠粪便中的脱落时程明显高于野毒株 ,P <0 .0 1。〔结论〕O157Sakai中的yhiE基因可能具有调节O157粘附肠道上皮细胞能力的作用 ,尽管其调节机制尚有待于进一步阐明 ,但本文结果显示其可能具有负调节的作用。另外 。

〔Objective〕 To construct a mutant strain with increased adherence to caco-2 cells in enterohemorrhagic Escherichia coli O 157 :H 7 (EHEC O 157 :H 7) to provide feasible measures for its in vivo and in vitro studies.〔Methods〕 The bacteria collected from an EHEC O 157 :H 7 strain mutagenized by mini-Tn5Km2 were screened fro their ability to increase the number of microcolonies (MC) on caco-2 cells and 8 mutants with increased adherence were isolated.〔Results〕 Analysis of the mini-Tn5Km2-flanked DNA sequences indicated that 6 mutants had their insertions in the yhiE gene.When 1 of the yhiE insertion mutants was orally inoculated into ICR mice,the number of bacteria shed into feces on day 14 was greater than that for the wild type.〔Conclusions〕 yhiE and yhiF are involved in the adherence of O 157 Sakai to epithelial cells.