Construction and preliminary study of biological functions of O157∶H7 typeⅢsecre-tion system effector NleF gene knockout mutant
10.7644/j.issn.1674-9960.2015.10.004
- VernacularTitle:肠出血性大肠杆菌O157∶H7Ⅲ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
- Author:
Tingting XU
;
Ting SONG
;
Wei ZHOU
;
Hongmei DAI
;
Junjie YUE
;
Long LIANG
- Publication Type:Journal Article
- Keywords:
enterohemorrhagic Escherichia coli;
O157∶H7;
NleF;
gene knockout;
T3SS;
λ-Red homologous recombina-tion
- From:
Military Medical Sciences
2015;(10):747-750
- CountryChina
- Language:Chinese
-
Abstract:
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 ΔnleF was constructed with λ-Red homologous recombination .Complementary strain ΔnleF/NleF was constructed by transferring pET-24a(+)-NleF into ΔnleF competent cells.Wild type,ΔnleF and ΔnleF/NleF were cultured in LB and DMEM(10%FBS) respectively,D600 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 ΔnleF and ΔnleF/NleF were constructed .The growth rates of wild type , ΔnleF and ΔnleF/NleF was not significantly different .Wild type O157 infection induced cell death .Cytotoxicity was increased as much in ΔnleF in-fected cells as in ΔnleF/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 .
