痢疾杆菌酸抗性系统相关基因缺失突变体的构建

Construction of Deletion Mutant of Shigella flexneri Acid Resistance Genes

依赖于谷氨酸脱羧酶的酸抗性系统对痢疾杆菌在宿主细胞内的生存和繁殖至关重要,hdeA、hdeB、yhiE和yhiF是其中几个重要的酸抗性基因。借助Red系统的重组功能,PCR扩增两翼与目的基因上下游同源的抗性基因片段,电击转化痢疾杆菌2457T,对筛选到的阳性转化子再导入编码FLP位点特异性重组酶的质粒pCP20以去除抗性基因。结果成功的敲除了hdeA、hdeB、yhiE和yhiF等4个酸抗性系统相关基因,为深入研究痢疾杆菌酸抗性基因的调控网络奠定了基础。

Glutamate-dependent acid resistance system is essential for successful colonization of the mammalian host by Shigella flexneri. Several genes such as hdeA, hdeB, yhiE and yhiF are very important to acid resistance. With the help of Red recombinant system, the kanamycin resistant gene flanked by homologues of target genes was amplified by PCR. The PCR products were electro-transformed into S.flexneri 2a strain 2457T. After target genes were replaced by the kanamycin gene, the resistance gene was then eliminated by using pCP20, a helper plasmid encoding the FLP recombinase. At last, four mutants of S.flexneri 2a strain 2457T, from which hdeA, hdeB, yhiE and yhiF gene were completely deleted, were successgully constructed by this procedure. This work provides the basis for elucidating the regulatory network of acid resistance genes.