肠出血性大肠杆菌O157:H7△hly△stx△toxB基因缺失株的构建

摘要

Enterohemorrhagic Escherichia coli 0157: H7 is a foodborne and zoonotic pathogen. Shiga toxin (Stx) , haemolysin ( Hly) and ToxB are the major virulent factors of 0157;H7. Therefore, the objective of this study is to construct the stx, hly ,toxB-knockout strain for develop 0157:H7 attenuated vaccine. The 0157 : 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 0157: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 Ka-namycin gene cassettes by allelic exchanges. Also,the 0157:H7(△hly △stx △toxB ) was found to be no differences compared with the parent 0157:H7 in growth feature. For Balb/c mice, the fecal shedding of 0157:H7 ( △toxB) was only 1.1×105 CFU on the third day after oral infection, whereas the parent 0157:H7 was 4. 03×107 CFU. Al-so,the mice infected with O157:H7(△hly △stx △toxB ) shed the bacteria for 9 days, 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.%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.