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目的构建肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC)O157∶H7 T3SS效应蛋白Nle F敲除菌株和回复菌株,研究其对细菌生长和细胞死亡的影响。方法利用λ-Red同源重组的方法构建nle F基因敲除菌株Δnle F;将p ET-24a(+)-Nle F重组质粒导入敲除菌感受态细胞中构建回复菌株Δnle F/Nle F。将野生株、敲除株和回复株分别用LB和DMEM(10%FBS)培养,每隔1 h测定D600,绘制生长曲线;分别用3种菌株感染He La细胞,用细胞毒性试剂盒检测上清中乳酸脱氢酶(LDH)的释放量,计算细胞毒性。结果成功构建Nle F敲除菌株Δnle F和回复菌株Δnle F/Nle F;野生株、敲除株和回复株三者的生长速率无明显差异;与野生株相比,Δnle F感染He La细胞后,细胞毒性增加,Δnle F/Nle F感染后He La细胞毒性与野生株相当。结论 EHEC O157∶H7 T3SS效应蛋白Nle F对细菌的生长无明显影响,但可能抑制由细菌感染引起的宿主细胞死亡。
Objective To construct Enterohemorrhagic Escherichia coli (EHEC) O157: H7 T3SS effector protein Nle F knockout and recovery strains and study their effects on bacterial growth and cell death. Methods The nle F gene knockout strain Δnle F was constructed by λ-Red homologous recombination. The recombinant plasmid pET-24a (+) - Nle F was introduced into the knockout competent cells to construct the retrovirus strain Δnle F / Nle F. The wild type, knockout and recovery strains were respectively cultured in LB and DMEM (10% FBS), the D600 was measured every 1 hour, and the growth curve was drawn. He La cells were infected with 3 strains respectively and detected by cytotoxicity kit Qing lactate dehydrogenase (LDH) release, calculate cytotoxicity. Results The Nle F knockout strain Δnle F and the recombinant strain Δnle F / Nle F were constructed successfully. The growth rate of wild type, knockout and recovered strains was no significant difference. Compared with the wild strain, Δnle F was infected by He La cells , Cytotoxicity increased, and He La cytotoxicity was comparable to wild-type strain after infection with Δnle F / Nle F. Conclusion The EHEC O157: H7 T3SS effector Nle F has no obvious effect on the growth of bacteria, but may inhibit the death of host cells caused by bacterial infection.