志贺菌毒力大质粒大片段敲除方法的建立

A Knocking-Out Method of Large Fragment of Virulence Plasmid of Shigella spp.

目的:构建志贺菌毒力大质粒大片段缺失突变体库。方法:首先利用λ-Red重组系统构建弗氏2a志贺菌301株毒力大质粒特定位点缺失株,再在距离此位点20 kb处缺失另一突变位点,最后根据重组酶识别远端FRT位点的特性,将两个远端FRT位点之间的DNA序列全部缺失。结果:敲除了毒力大质粒24 kb的DNA序列。结论:利用λ-Red重组系统及FLP-FRT位点特异性识别重组系统可以对志贺菌毒力大质粒逐步进行大片段的敲除,构建大质粒大片段缺失突变体库。

Objective：To construct a library of large fragment of virulence plasmid deletion mutants of Shigella spp.Methods：Firstly,using λ-Red recombination system,we knocked out the first fragment of large virulence plasmid;then we knocked out another fragment of 20 kb away;finally using the characteristics that recombinase can recognize the far FRT,we deleted the DNA fragment between the two FRT.Results：we successfully knocked out a large fragment about 24 kb from virulence plasmid of Shigella spp.Conclusion：A library of virulence plasmid deletion mutants could be constructed through gradually deleting large fragments of the virulence plasmid of Shigella spp.by λ-Red recombination system and the FLP-FRT site-specific recombination system.