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肠出血性大肠杆菌O157∶H7 Ⅲ型分泌系统效应蛋白NleF基因敲除菌株的构建及其生物学功能初探

Construction and preliminary study of biological functions of O157∶H7 type Ⅲ secretion system effector NleF gene knockout mutant
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摘要 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 Escherichia coli O157:H7 T3SS effector NleF gene knockout mutant and its com- plementary strain, and probe its effects on bacterial growth and cell death. Methods T3SS Effector NleF gene knockout mutant AnleF was constructed with λ-Red homologous recombination. Complementary strain AnleF/NleF was constructed by transferring pET-24a( + )-NleF into AnleF competent cells. Wild type, AnleF and AnleF/NleF were cultured in LB and DMEM( 10% FBS) respectively,D was measured every hour, and the growth curve was drawn. HeLa cells were infected with three kinds of strains, the supernatant of LDH release was detected with cytotoxicity detection kit, and the cytotoxicity was calculated. Results /tnleF and AnleF/NleF were constructed. The growth rates of wild type, ΔnleF and AnleF/NleF was not significantly different. Wild type O157 infection induced cell death. Cytotoxicity was increased as much in AnleF infected cells as in AnleF/NleF infected cells. Conclusion EHEC O157:H7 T3SS Effector NleF has no significant effect on bacterial growth, but might inhibit host cell death caused by bacterial infection.
出处 《军事医学》 CAS CSCD 北大核心 2015年第10期747-750,共4页 Military Medical Sciences
基金 国家自然科学基金资助项目(31400046)
关键词 肠出血性大肠杆菌 O157∶H7 NleF 基因敲除 T3SS λ-Red同源重组 enterohemorrhagic Escherichia coli 0157: H7 NleF gene knockout T3SS λ-Red homologous recombination
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