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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。
Using λ-Red recombination system, the ipaH4.5 gene of 301 strains of Shigella flexneri was deleted and mutated, and 301 strains of ipaH4.5 gene deletion mutant of Shigella flexneri was constructed. Plasmid construction ipaH4.5 deletion mutant of the mutant iΔHpaH4.5HF. PCR method confirmed the ipaH4.5 gene deletion and response. The growth, metabolism and cell invasion ability of wild strain, mutant strain and replicative mutant strain were compared. The levels of inflammatory cytokines in the supernatant of J774 macrophages were detected by ELISA. Growth and metabolism experiments showed that the deletion and recovery ipaH4.5 did not affect the growth rate of Shigella invasion experiments showed that ipaH4.5 deletion and recovery also did not affect Shigella invasion of HeLa cells and murine J774 macrophages, The ipaH4.5 gene has nothing to do with the growth, metabolism and invasion ability of Shigella. The changes of cytokines in the supernatant of J774 macrophages suggest that the gene inhibits the host cell inflammatory response after Shigella invasion.