Characterization of DegQVh, a Serine Protease and a Protective Immunogen from a Pathogenic Vibrio harveyi Strain
Characterization of DegQVh, a Serine Protease and a Protective Immunogen from a Pathogenic Vibrio harveyi Strain
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DOI：
10.1128/aem.00109-08
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发表时间：
2008-08
影响因子：
4.4
通讯作者：
Wei-Wei Zhang-Wei;Kun Sun;Shuang Cheng;Li Sun
中科院分区：
文献类型：
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作者：
Wei-Wei Zhang-Wei;Kun Sun;Shuang Cheng;Li Sun
摘要哈维氏弧菌是一种重要的海洋病原菌，可感染多种水产养殖动物。哈维氏弧菌（V. harveyi）degQ（degQVh）是编码DegQ同源物的基因，其从从病鱼分离的致病性哈维氏弧菌菌株T4中克隆。DegQVh与其他弧菌属中鉴定的HtrA家族成员密切相关，并且可以补充degP缺陷的大肠杆菌菌株的温度敏感表型。T4中degQVh的表达受温度调节，可能通过σ E样因子。酶切分析表明，重组DegQVh蛋白在E.大肠杆菌是一种活性丝氨酸蛋白酶，其活性需要催化位点和PDZ结构域的完整性。重组DegQVh蛋白的最适温度和pH为50°C和pH 8.0。疫苗接种研究表明，纯化的重组DegQVh是一种保护性免疫原，可以保护鱼类免受哈维氏弧菌的感染。为了提高DegQVh作为疫苗的效率，构建了质粒pAQ 1形式的遗传构建体，其中编码经加工的DegQVh蛋白的DNA与编码AgaV（一种胞外β-琼胶酶）分泌区的DNA融合。急诊重组质粒pAQ 1在大肠杆菌中表达，并分泌到培养上清中。用活的E.表达嵌合degQVh的大肠杆菌显著（P < 0.001）提高了鱼对哈维氏弧菌攻击的存活率，这可能是由于免疫系统相对延长地暴露于由载体菌株组成性产生的重组抗原，尽管水平逐渐降低。
ABSTRACT Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQVh), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQVh was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQVh in T4 was modulated by temperature, possibly through the σE-like factor. Enzymatic analyses demonstrated that the recombinant DegQVh protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQVh protein were 50°C and pH 8.0. A vaccination study indicated that the purified recombinant DegQVh was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQVh as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQVh protein was fused with the DNA encoding the secretion region of AgaV, an extracellular β-agarase. The E. coli strain harboring pAQ1 could express and secrete the chimeric DegQVh protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQVh significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.