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目的:检测n espO基因敲除对肠出血性大肠埃希菌(EHEC)生物学特性的影响。n 方法:自杀质粒pCVD442-Δn espO介导的两步法构建n espO基因敲除菌株(Δn espO)，pTrc99a质粒构建n espO基因回补突变株(CΔn espO)，不同EHEC菌株感染HeLa细胞以了解n espO基因在感染过程中的生物学功能与致死作用。n 结果:PCR和电泳以及基因测序结果表明，Δn espO和CΔn espO突变株构建成功。与野生株相比，Δn espO和CΔn espO突变株的生长速度无明显差异，表明n espO基因不影响细菌的生长与繁殖。此外，EspO可激活肿瘤坏死因子(TNF)诱导的NF-κB信号通路，而效应蛋白NleB能够抑制该激活过程。在细菌感染细胞试验中，EspO不能抑制TNF和TRAIL(TNF-related apoptosis-inducing ligand)诱导的细胞死亡。n 结论:本研究成功构建了EHEC n espO基因敲除和回补突变株菌株，初步探究了n espO与宿主细胞的相互作用及该基因在感染过程中对细胞凋亡的影响，为后续深入研究细菌EspO蛋白的体外生物学活性以及体内感染过程中的致病作用提供实验依据。n “,”Objective:To analyze the effects of n espO gene knockout on the biological characteristics of enterhemorrhagic n Escherichia coli (EHEC).n Methods:Two-step methods mediated by the suicide plasmid pCVD442-Δn espO and plasmid pTrc99a were used to construct the n espO gene-deleted strain (Δn espO) and the complemented mutant (CΔn espO), respectively. HeLa cells were infected with different EHEC strains to analyze the biological functions and lethal effects of n espO gene during infection.n Results:PCR, electrophoresis and gene sequencing showed that the Δ n espO and CΔn espO mutants were successfully constructed. Compared with the wild-type strain, neither the Δ n espO nor CΔn espO mutant showed significant difference in growth rate, indicating that the n espO gene had no influence on the growth and replication of EHEC. Furthermore, EspO could activate the tumor necrosis factor receptor (TNF)-induced NF-κB signaling pathway, while the effector protein NleB could inhibit the process. EspO could not inhibit the death of HeLa cells induced by TNF or TNF-related apoptosis-inducing ligand (TRAIL) after EHEC infection.n Conclusions:In this study, we successfully constructed the n espO gene-deleted and complemented mutants of EHEC and preliminarily analyzed the interaction between n espO gene and host cells and the effects of n espO gene on cell apoptosis during infection, which provided reference for further research on the n in vitro biochemical activity and n in vivo pathogenic roles of EspO.n