Construction and Identification of Enterohemorrhagic Escherichia coli O157:H7 Deficient in Stx,Hly and ToxB Expression

doi:10.7668/hbnxb.2011.05.016

Abstract

Abstract: Enterohemorrhagic Escherichia coli O157:H7 is a foodborne and zoonotic pathogen.Shiga toxin(Stx),haemolysin(Hly)and ToxB are the major virulent factors of O157:H7.Therefore,the objective of this study is to construct the stx,hly,toxB-knockout strain for develop O157:H7 attenuated vaccine.The O157:H7(△hly△stx△toxB)was constructed by choosing the sm10 complement cells and the suicide plasmid pMEG375 containing homologous arms of stx,hly and toxB upward and downward genes to study their effect on O157:H7 intestinal colonization of streptomycin-treated mice and adherence to HEp-2 cells.The results from PCR and Western blotting methods showed that stx,hly and toxB genes were successfully replaced with the Gentamicin,Spectinomycin and Kanamycin gene cassettes by allelic exchanges.Also,the O157:H7(△hly△stx△toxB)was found to be no differences compared with the parent O157:H7 in growth feature.For Balb/c mice,the fecal shedding of O157:H7(△toxB)was only 1.1×105 CFU on the third day after oral infection,whereas the parent O157:H7 was 4.03×107 CFU.Also,the mice infected with O157:H7(△hly△stx△toxB)shed the bacteria for 9days,whereas the parent O157:H7 for above 14 days.O157:H7(△hly△stx△toxB)successfully constructed in this study was found to be attenuated in HEp-2 cells of adherence for four times and in mouse models of colonization for shortened and lower fecal shedding in contrast with the parent O157:H7.

Key words: Enterohemorrhagic Escherichia coli O157:H7, hly, stx, toxB gene, Knockout strain, Mouse Adherence

CLC Number:

S852.61

ZHANG Xue-han, HE Kong-wang, ZHAO Pan-deng, LUAN Xiao-ting, YE Qing, WEN Li-bin, NI Yan-xiu, ZHOU Jun-ming, LI Bin, WANG Xiao-min, GUO Rong-li, YU Zheng-yu, MAO Ai-hua, LU Li-xin. Construction and Identification of Enterohemorrhagic Escherichia coli O157:H7 Deficient in Stx,Hly and ToxB Expression[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(5): 76-82. doi: 10.7668/hbnxb.2011.05.016.

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