CAJ | 학술논문

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。
목적：구건O157∶H7 EDL933균주Ⅲ형분비계통（ T3SS）결함돌변주ΔescR，감염HeLa세포후검측기점부능력。방법통과중첩연신PCR확증출중간위잡나매소항성기인，량측위escR기인상하유동원서렬적중조선성DNA편단，전격전입함pKD46적EDL933감수태세포중，escR기인피kan기인체환이결실。회제야생주여ΔescR생장곡선，병이용면역형광관찰량주세균대HeLa세포적점부능력。결과획득결함돌변주ΔescR，상교우야생주，해돌변주재DMEM（10％FBS）배양기중생장속도이급대HeLa세포적점부능력명현하강。결론 T3SS결구단백escR기인적결실파배료EDL933 T3SS적형성，영향료해균주점부능력，기구건위후속연구T3SS전정료기출。
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.