【摘要】 目的 E.tarda能够在体内外许多环境包括在巨噬细胞内生存和繁殖,eha基因是该菌一个重要的转录调控基因,本研究探讨该基因在细菌抵抗巨噬细胞内外压力中的作用及机制。方法采用体外实验模拟巨噬细胞内氧化和酸杀菌条件、体内血清和胆汁杀菌条件、以及细菌对SDS和多粘菌素B敏感性试验,比较ET-13毒力株及其Δeha缺失株和ehaComp互补株在这些压力下存活率,利用RT-PCR和SDS-PAGE电泳,比较上述3种细菌相关基因的转录和表达的差异。结果eha基因的缺失使ET-13对酸、H2O2、SDS和多粘菌素B敏感性提高(P<0.05);经小鼠血清或鱼胆汁处理后,3种菌株的存活率没有明显区别(P>0.05);RT-PCR结果显示,eha基因的缺失使得E.tarda内的超氧化物歧化酶基因sodC、过氧化氢酶基因katB和鞭毛蛋白基因fliC、Ⅲ型分泌系统分泌蛋白基因eseC的转录水平下降。SDS-PAGE电泳结果显示,缺失株的主要外膜蛋白比野生株表达降低。结论 eha基因通过调控E.tarda相关基因的表达,参与了该菌抵抗巨噬细胞内氧化和酸等的压力,有助于细菌在巨噬细胞内生存和繁殖。更多还原

【Abstract】 Edwardsiella tarda(E.tarda)could exist and replicate in vivo and in vitro such as in macrophage.The eha gene is an important regulating gene in E.tarda.We explored its role and mechanism for E.tardato resist to macrophage stresses in this study.We compared the survival rates of ET-13virulence strains with their eha mutant strains(Δeha)and complementary strains(ehaComp)by a series of stress experiments in vitro,which were imitated oxidation and acid condition in macrophage,serum and bile condition in vivo.Compared with the wilds,the survival rates of eha mutant strains decreased significantly(P<0.05)in the conditions of acid,H2O2,SDS,and polymyxin B.There’s no significant difference(P>0.05)between the survival rates of eha mutants and ones of the wild strains with the treatment of mice serum or fish bile.The transcription level of the genes of superoxide dismutase C(sod C),catalase B(katB),flagellin C(fliC),and secretary protein C(eseC)of III type secretary system protein in the mutants were lower by RT-PCR.The expression of main outer membranes in the mutants was decreased by SDS-PAGE electrophoresis.The results showed that the ehagene may control some related virulence factors for E.tardato resist acid and oxidants to kill in macrophage.The ehagene was helpful for E.tardato replicate in macrophage.更多还原