利用λRed重组系统敲除伤寒沙门氏菌rfaH基因

目的：利用λRed重组系统敲除伤寒沙门氏菌的rfaH基因。方法：以伤寒沙门氏菌（Salmonella typhi Ty2,S.ty2）基因组为模板扩增得到的同源臂,与两端带有FRT位点的卡那霉素抗性基因片段共同构建同源重组载体;以重组载体为模板扩增打靶片段,将其转化S.ty2;在抗生素压力和λRed重组系统帮助下,打靶片段和菌体基因组发生同源重组,通过卡那抗性筛选得到带有抗性标记的重组菌;转入重组酶表达质粒pCP20以去除抗性标记,得到保留单一FRT位点的突变菌株;通过PCR鉴定重组菌,并经透射电子显微镜分析表型。结果：在S.ty2中敲除了rfaH基因,经PCR扩增和序列测定正确;初步的表型分析表明突变体的鞭毛合成显著减少。结论：获得了S.ty2突变株,为将沙门氏菌进一步减毒成为疫苗表达载体奠定了基础。

Knock-Out of rfaH Gene in Salmonella typhi Ty2 by λRed Recombination System

Objective： To construct rfaH gene deletion mutant of Salmonella typhi Ty2（S.ty2） with λRed recombination system. Methods： Homologous regions and kanamycin cassette with two FRT sites were amplified from the corresponding templates and were inserted into plasmid pET22b-kana to construct a recombinant vector. The resultant fragments were then amplified from the recombinant vector and transformed into S.ty2. Under the pressure of antibiotic and with the work of λRed system, homologous recombination occurred between the fragments and genome of host strain, and the recombinants were selected on kanamycin agar plate. Plasmid pCP20 was introduced into the recombinant to remove the kanamycin resistant relevant DNA fragment, resulting in a single FRT site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR. Phenotype analysis was performed by transmission electron micrographs. Results： rfaH gene deletion mutant was obtained and identified by PCR method and sequencing. Phenotype analysis indicated that the flagella synthesis of mutant decreased dramatically. Conclusions： The rfaH deletion mutant of Salmonella typhi Ty2 was obtained, and it will be modified further to be the live attenuated bacterial vector.