志贺氏菌福氏2a 301株ipaH4.5突变株的构建及生物学功能分析

Construction and Function Analysis of the ipaH4.5 Gene Deletion Mutant of Shigella flexneri 2a Strain 301

利用λ-Red重组系统对福氏2a志贺氏菌301株ipaH4.5基因进行缺失突变,构建了福氏2a志贺氏菌301株ipaH4.5基因缺失突变株?ipaH4.5,利用低拷贝质粒构建ipaH4.5缺失突变株的回复突变株△ipaH4.5HF。PCR方法证实了ipaH4.5基因的缺失和回复。对野生株、突变株和回复突变株的生长代谢及细胞侵袭能力进行比较;ELISA方法检测3株菌侵袭鼠J774巨噬细胞后培养上清中炎性因子的水平。生长代谢实验表明缺失和回复ipaH4.5不影响志贺氏菌的生长速度,侵袭实验表明缺失和回复ipaH4.5也不影响志贺氏菌对HeLa细胞和鼠J774巨噬细胞的侵袭能力,表明ipaH4.5基因与志贺氏菌的生长代谢和侵袭能力无关;鼠J774巨噬细胞培养上清中细胞因子水平的改变提示该基因在志贺氏菌侵入细胞后抑制宿主细胞炎症反应。

A γ-Red recombination system was used to construct an ipaH4.5 deletion mutant of S. flexneri 2a 301. A recovery mutant was obtained by introducing a low-copy plasmid strain. PCR analysis confirmed that ipaH4.5 was successfully deleted in the mutant and restored in the complementary strain. The growth curves of wild-type, deletion mutant and recover mutant were measured. Some biochemical tests were investigated. Invasion tests were performed to evaluate the virulence of these three strains. Quantities of cytokines in the culture supernatants of murine macrophages cell line J774 after being infected was measured by enzyme-linked immunosorbent assays （ELISA）. No significant difference of growth rates and basic biochemical events were observed among three strains, and no difference in invasion ability was observed either. However, More IL-1β and TNF-а were observed in murine macrophages infected with deletion mutant than wild type and recover mutant. So we conclude that ipaH4.5 can inhibit inflammation response of the host after Shigella flexneri 2a 301 enter into the cell.