| Edwardsiella tarda is an important pathogenic bacterium in aquaculture, so its immune protection is of significance. In this study, 9 potential protective proteins of E. tarda were analyzed for the effect of immune protection by using Japanese flounder as animal models. The results showed that two of these proteins, EseD and Et18, exhibited significant protection. For the improvement of their protective efficiency, the two antigens were fused together via genentic engineering techniques and resulted in a hybrid protein EEH. Vaccination results revealed that the protective efficiency of EEH is higher than both EseD and Et18. ELISA and Western blotting analysis showed that the three recombinant proteins caused the production of significant levels of specific antibodies in Japanese flounder. This study provides theoretical basis for the development of E. tarda vaccines.In addition, the E. tarda AcrAB multidrug resistance system was cloned in this study. Sited-directed mutation was used to determine the promoter sequences of acrAB and acrR and the AcrR binding site. Promoter activity analysis demonstrated that the presence of AcrR casued 300-fold reduction in the activity of the acrAB promoter and 3-fold reduction in the activity of the acrR promoter. Sited-directed mutations showed that K39 and R45 are important for AcrR activity; deletion analysis results revealed that the N terminal 205 amino acid residues are essential to the function of AcrR. Acriflavine, Ethidium Bromide, Methyl Viologen and Sodium Dodecyl Sulfate are found to be the inductors of AcrR. Analysis of the acrR-overexpressing strain demonstrated that its drug resistance level, growth condition and virulence are lower than the control group. This study improves the understanding of multidrug resistance mechanism of E. tarda as well as its relevance to virulence. |
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