目的：构建O157∶H7 EDL933菌株Ⅲ型分泌系统（ T3SS）缺陷突变株ΔescR，感染HeLa细胞后检测其黏附能力。方法通过重叠延伸PCR扩增出中间为卡那霉素抗性基因，两侧为escR基因上下游同源序列的重组线性DNA片段，电击转入含pKD46的EDL933感受态细胞中，escR基因被kan基因替换而缺失。绘制野生株与ΔescR生长曲线，并利用免疫荧光观察两株细菌对HeLa细胞的黏附能力。结果获得缺陷突变株ΔescR，相较于野生株，该突变株在DMEM（10％FBS）培养基中生长速度以及对HeLa细胞的黏附能力明显下降。结论 T3SS结构蛋白escR基因的缺失破坏了EDL933 T3SS的形成，影响了该菌株黏附能力，其构建为后续研究T3SS奠定了基础。“,”Objective To construct the typeⅢ secretion system (T3SS) deficient mutant of O157∶H7 EDL933, and to detect its ability of attachment after infection with HeLa cells.Methods The recombinant DNA fragments obtained kana-mycin resistant gene were constructed by overlap extension PCR and transferred into EDL933/pKD46 to replace escR gene by homologous recombination.Results The T3SS-deficient mutant (ΔescR) was successfully construted.Compared with the wild-type EDL933, the growth rate and attachment on HeLa cells of ΔescR were significantly decreased.Conclusion The deletion of escR gene, which encodes T3SS structural protein EscR, affects the attachment of EDL933, suggesting a better basis for future studies of T3SS.