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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。
Acid-dependent systems that rely on glutamate decarboxylase are crucial for the survival and reproduction of dysentery bacteria in host cells, and hdeA, hdeB, yhiE and yhiF are some of the important acid-resistance genes. With the recombination function of Red system, PCR-amplified resistance gene fragments with homology between upstream and downstream of the target gene were amplified by electroporation and transformed into Shigella dysenteriae 2457T. The positive transformants were introduced into plasmid pCP20 encoding FLP site-specific recombinase To remove the resistance gene. Results Successful knockout of 4 acid resistance genes related to hdeA, hdeB, yhiE and yhiF laid the foundation for further study on the regulatory network of Shigella acid resistance genes.