肠出血性大肠杆菌O157∶H7 toxB基因缺失株的构建

摘要

In order to definite the correlation between ToxB, a large protein encoded by pO157 of enterohemorrhagic Escherichia coli 0157 : H7, and intestinal adherence and colonization of 0157 : H7, the recombinant suicide plasmid pMEG375-BN-Kan-BC was construted by cloning homologous arms of loxB upward and downward genes into suicide plasmid, and was transformed into host bacterium SM10 complement cells to obtain recombinant SM10(pMEG375-BN-Kan-BC). The SM10(pMEG375-BN-Kan-BC) was incubated with 0157 : H7 to construct 0157 : H7( △toxB) tluough homologous recombination. HEp-2 cells inoculated in vitro and streptomycin-treated mice in vivo were used to identify 0157 : H7 ( △toxB) adherence and colonization. The results from PCR and Western blotting showed that toxB gene was successfullyrnreplaced with the kanamycin gene cassette by an allelic exchange. No differences were found between the 0157 : H7 ( AtoxB) and the parent 0157 : H7 in growth feature. 0157 : H7 ( △toxB) was two-times attenuated in adherence to HEp-2 cells. For Balb/c mice, the fecal shedding of 0157 : H7 ( △toxB) was only 3. 1 × 105 CFU on the third day after oral infection, whereas the parent 0157 :H7 was 4.03×l07 CFU. The mice infected with 0157 : H7 ( △toxB) shed the bacteria for 11 d, while with the parent 0157 : H7, the mice shed for 14 d.%为了明确肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli)O157∶H7大质粒pO157编码的蛋白质ToxB与O157∶H7在肠道黏附、定殖的关系,构建含有toxB基因同源臂的重组自杀性质粒pMEG375 -BN-KanBC,转化SM10宿主菌获得重组SM10( pMEG375-BN-Kan-BC).将SM10(pMEG375-BN-Kan-BC)与O157∶H7进行混合培养,通过同源重组,获得杂交toxB基因缺失株.用体外接种的HEp-2细胞和体内感染链霉素处理的小鼠,明确缺失株黏附定殖能力.经PCR和Western blot鉴定,toxB基因被卡那霉素(Kan+)选择标记基因表达盒所代替.O157∶H7(△toxB生长特性与亲本株相比无明显差异.O157∶H7(△toxB)与亲本株相比,对HEp-2细胞的黏附能力降低了2倍.口服感染小鼠后,O157∶H7(△toxB第3d粪便排菌量仅为3.1×105 CFU,持续排菌11 d；而亲本株排菌量高达4.03×107 CFU,持续排菌时间超过14 d.