eha,迟缓爱德华菌一个毒力调控基因

eha,a regulating virulence gene of Edwardsiella tarda

目的探讨迟缓爱德华菌(Edwardsiella tarda,E.tarda)溶血相关基因(E.tardahaemolysin activator gene,eha)基因调控该菌毒力基因的作用。方法利用自杀质粒pHM5,缺失E.tarda的eha基因,得到△eha缺失菌株,再构建△eha株的互补菌株。通过平板溶血性法、接触溶血法、上清溶血法,观察野生株、△eha缺失株与及互补株溶血性的差异。利用MTT法比较三种细菌培养物的过滤液对Vero细胞的毒性的差别。利用H2O2抗性纸片扩散法,比较三种菌对过氧化氢抵抗力的差异。利用RT-PCR和SDD-PAGE电泳超速酸化离心法提取的鞭毛蛋白,比较三种细菌鞭毛基因的转录和表达的差异。结果△eha缺失株和互补株不溶血,而野生株溶血。eha基因的缺失降低E.tarda菌对过氧化氢的抵抗力,△eha缺失株较野生株的细胞毒性明显减弱,eha基因可以调控迟缓爱德菌鞭毛基因的转录和表达。结论 eha基因可以调控迟缓爱德华菌毒力基因的表达,是一个毒力调控基因。

To explore the function of eha gene on regulating E.tarda virulence genes which was deleted by suicide plasmid pHM5 from E.tarda.The △eha mutant strain was obtained and the △eha complementary strain was constructed.Differences among the wild strains,mutant strains and complementary strains were compared by plate hemolysis,contact hemolysis and supernatant assay,respectively.The filtering cultures of the three kinds of E.tarda were used to test vero cell cytotoxicity with MTT method.Comparisons of the three kinds of E.tarda against hydrogen peroxide were conducted by scrip diffusion and the bacterial flagellin protein was purified by acidification excessive speed centrifugation.The transcription and expression on the bacterial flagellin gene of them were also compared by RT-PCR and SDS-PAGE electrophoresis.Results indicated that the mutant and complementary strains were non-hemolytic,while the wild strains were hemolytic.E.tarda decreased its resistance against hydrogen peroxide due to deletion eha and mutant strains produced less cytotoxicity than the wild ones.The transcription and expression of flagellin gene were regulated by eha gene.It＇s concluded that eha gene plays a regulatory role on the expression of virulence gene of E.tarda and the eha gene was a regulating virulence gene of E.tarda.