Expression of Vibrio harveyi Hemolysin Gene vhhA in E.coli and its Biological Activities

Vibrio harveyi is an important pathogen in cultured fish and shrimp.Previously,it was found that the hemolytic activity of the extracellular product(ECP) of V.harveyi was involved in pathogenesis in salmonids,and the most pathogenic isolate,VIB 645,contains two closely related hemolysin genes,i.e.vhhA and vhhB.In this study,vhhA was cloned into an expression plasmid pET-24d(+),and the recombinant hemolysin protein was overexpressed in Escherichia coli overexpression strain BL21(DE3) as a His-tag fused protein.The recombinant E.coli revealed a strong hemolytic activity on fish blood agar plate and phospholipase activity on egg yolk emulsion plate.The molecular weight of the expressed hemolysin was about 43 kDa analyzed by SDS-PAGE.The recombinant hemolysin can be induced to express at 17,25 and 37 ℃,and the times to obtain the highest quantities of protein were 9,6 and 3 h, respectively.Also,there were more expressed hemolysins secreted into the culture medium when the recombinant E.coli was induced at 25 ℃.