摘要： 肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli)O157:H7是食源感染的人兽共患病原菌。志贺毒素(Shiga toxin,Stx)、溶血素(haemolysin,Hly)和ToxB是O157:H7重要的毒力因子。选用自杀性质粒pMEG375,体外构建具有同源臂的重组自杀性质粒,借助于sm10宿主菌获得O157:H7杂交菌株,通过同源重组,依次获得O157:H7(△hly△stx△toxB)基因缺失株,△hly△stx△toxB接种HEp-2细胞和感染链霉素处理的Balb/c小鼠,明确缺失株粘附定植的能力。经PCR鉴定,hly、stx和toxB基因分别被壮观霉素(Spc+)、庆大霉素(Gm+)和卡那霉素(Kan+)基因表达盒所代替,Western blot鉴定结果显示,O157:H7(△hly△stx△toxB)检测不到相应的Stx、Hly和ToxB蛋白。O157:H7(△hly△stx△toxB)生长特性与亲本株无明显差异。对于HEp-2细胞的粘附能力明显降低。Balb/c小鼠排菌时间和排菌量明显减少和降低。本研究成功获得O157:H7(△hly△stx△toxB),并且明确Stx、Hly和ToxB的缺失导致了O157:H7对HEp-2细胞和Balb/c小鼠的粘附和定植能力降低。本研究旨在为研制EHEC O157:H7基因缺失疫苗奠定物质基础。

关键词: 肠出血性大肠杆菌O157:H7, hly, stx, toxB, 缺失株, 小鼠, 粘附

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

中图分类号: 

• S852.61