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目的:构建对肠上皮细胞粘附缺陷的肠出血性大肠埃希菌O157∶H7(EHEC O157)突变株,为体内、体外研究其粘附机制提供可用工具。方法:将pSC101和m in i-Tn5Km2分别通过转化和结合导入O157Sakai菌株中,筛选粘附力缺陷的转化子感染Caco-2细胞株,观察Caco-2单层细胞中的微菌落数量和m in i-Tn5Km2插入位点。结果:经诱变,共分离到3组粘附缺陷突变株,1组完全丧失了粘附力,2组粘附力减弱,3组的粘附力更弱。1组和3组拥有LEE岛上多基因的转座子插入,而2组m in i-Tn5Km2的插入位点在LEE岛之外。结论:O157Sakai的肠道粘附能力可能与LEE岛的III型分泌系统有关。
Objective: To construct enterohemorrhagic Escherichia coli O157: H7 (EHEC O157) mutant with defective adhesion to intestinal epithelial cells and to provide a useful tool for studying its adhesion mechanism in vitro and in vivo. Methods: The Caco-2 cell lines were infected with transformants of O157Sakai through transformation and combination with pSC101 and m in i-Tn5Km2, respectively. The numbers of microcolonies and m in i-Tn5Km2 insertion site. Results: Three groups of mutant strains with adhesion defects were isolated. One group lost the adhesion completely, the two groups had weaker adhesion and the three groups had weaker adhesion. Groups 1 and 3 had a transposon insertion with multiple genes on the LEE island, while two sets of insertion sites for m in i-Tn5Km2 were outside the LEE island. Conclusion: The intestinal adhesion of O157Sakai may be related to the type III secretion system of LEE island.