Molecular Cloning, Expression And Functional Analysis HSPs From Edwardsiella Tarda

According to the reported genome sequence of Eedawarsiella tarda strain EIB202, dnaJ,dnaK and htpG gene were cloned from E. tarda strain TXOl,which was issolated from an infected turbot. Each of these gene was expressed in Escherichia coli strain BL21(DE3), and the recombinant protein were purified with nichel-nitrilotriacetic acid agarose under native conditions.The ATPase activity of DnaK and HtpG was measured by malachite green assay at different concentration of DnaK and HtpG,and the optimum temperature for HtpG was around 40℃. In the presence of DnaJ,the ATPase activity of DnaK was enhanced significantly.The regulatory role of DnaJ in the fuctioning of DnaK is assessed by pull down assay. The expression of those threee gene in TX01 were induced by higher temperature and H2O2.In order to investigate the functional importance of DnaJ and HtpG in the pathogenesis of E. tarda, dnaJ-defective E. tarda mutant and htpG-defective E. tarda mutant were constructed. Animal testing and cell experiments indicated that the mutants,retarded in growth and more sensitive to H2O2-induced oxidative damage,were severely vitiated in infectivity and in overall bacterial virulence.DnaJ and HtpG were tested to be immunoprotective protein agaist TX01.Both protein exhibited RPS(Relative Percentage Survival),62.2% and 53.1%,aganist the disease caused by TX01.