哈维氏弧菌溶血素基因vhhA在大肠杆菌中的表达及活性研究

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

哈维氏弧菌(Vibrio harveyi)是水产养殖动物(鱼、虾)的重要致病菌。已发现哈维氏弧菌对鱼类的致病性与其分泌的胞外产物中的溶血素相关,致病力最强的菌株VIB 645有2个碱基序列非常相似的溶血素基因,vhhA和vhhB。本研究将vhhA克隆于pET-24d(+)表达质粒,VHH溶血素蛋白作为1种带6组氨酸的融合蛋白在大肠杆菌BL21(DE3)中得到了过量表达。重组菌在鱼血平板上表现出很强的溶血活性,并且在卵磷脂平板上表现出磷脂酶活性。SDS-PAGE分析表明,表达蛋白的分子量约为43 kDa。在17,25和37℃时重组的VHH溶血素蛋白都能得到很好的表达,达到最大表达量的时间分别为9,6和3 h。在25℃进行诱导时,分泌到上清液中的表达蛋白的量最多。

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℃.