弗氏志贺菌 M90T 株毒力相关膜蛋白复合物组成基因缺失突变体的构建

目的：构建弗氏志贺菌M90T株毒力相关膜蛋白复合物组成蛋白基因的缺失突变株。方法：分别扩增目的基因的上、下游同源臂和卡那霉素抗性基因片段，利用重叠延伸PCR技术将这3个片段融合为打靶片段，电击转化M90T／pKD46感受态细胞，在bRed重组系统的作用下，通过同源重组将目的基因置换为卡那霉素抗性基因，之后在辅助质粒pCP20的作用下切除FRT位点之间的卡那霉素抗性基因，得到基因缺失突变体。结果与结论：分别构建了M90T株毒力相关膜蛋白复合物4个组成蛋白Apy、DnaK、CIpB、YdgA的基因缺失突变体，为进一步研究各基因的功能提供了突变体。

Construction of Gene Knock-Out Mutants of Components of a Virulence Related Membrane Protein Complex of Shigella flexneri M90T

Objective： To knock out components of related membrane protein complex of Shigella flexneri M90T coding gene respectively and obtain the corresponding mutant. Methods： DNA fragments composed of flank se- quences of target gene and kanamycin resistance gene between them were obtained by overlap extension PCR, and then transferred into M90T/pKD46 to replace target gene with kanamycin resistance gene by homologous recombina- tion with help of kRed recombination system. The kanamycin resistance gene between FRT sites was subsequently removed with help of plasmid pCP20. Results ＆ Conclusion： Four gene knock-out mutants of components Apy, DnaK, ClpB and YdgA of a virulence related membrane protein complex of M90T were successfully constructed. These mutants will be applied in the function studies of these genes.