肠出血性大肠杆菌O157:H7△hly△stx△toxB基因缺失株的构建

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

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。

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 smlO 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 （AtoxB） was only 1.1 ×l0s CFU on the third day after oral infection,whereas the parent O157 ：H7 was 4.03 × 107 CFU. A1so, the mice infected with 0157 ：H7 （ △ hly △ stx △ toxB）shed the bacteria for 9days, whereas the parent 0157 ： H7 for above 14 days. 0157 ： 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 0157 ：HT.