【作者】 杨朝；

【导师】 张元兴； Roy Curtiss Ⅲ；

【作者基本信息】 华东理工大学 ， 生物化工， 2010， 博士

【摘要】 鳗弧菌是一类可以在世界范围内引起淡水和海水鱼类疾病大爆发的重要病原菌。本实验室前期工作从鳗弧菌野生株MVM425出发,通过重组DNA技术得到一株减毒株,初步的免疫试验表明,MVAV6203有较大的潜力开发成为成功的减毒活菌疫苗。为了在鳗弧菌减毒活疫苗的基础上开发潜在的多价重组疫苗,将保护性抗原蛋白递呈到载体表面,需要引入高效的表面展示系统。本实验利用来源于鳗弧菌的外膜蛋白作为锚定元件,构建了一系列新型表面展示系统。该类系统的锚定元件由两部分组成：1)鳗弧菌外膜脂蛋白Wza信号肽及其N端的7个氨基酸或者大肠杆菌主要外膜脂蛋白Lpp信号肽以及N端的九个氨基酸；2)鳗弧菌外膜蛋白Omporfl, OmpU或Omp26La的跨膜区。将绿色荧光蛋白作为报告基因插入到这些系统中,构建的六个表面展示系统中有四个可以成功地将GFP展示到大肠杆菌细胞的表面；与Wza-Omporfl融合的来源于大黄鱼虹彩病毒的主要衣壳蛋白基因,也可以成功地展示到鳗弧菌减毒株MVAV6203的表面；通过将来源于迟钝爱德华氏菌的EseB抗原蛋白与六个展示系统融合,并转入MVAV6203中,可以将EseB展示到鳗弧菌表面,从而构建了一系列多价疫苗候选株。之后选用候选株AV/pW-26La-B,利用斑马鱼对其进行免疫保护评价,结果显示该菌株可以保护斑马鱼免受鳗弧菌感染,对迟钝爱德华氏菌感染的前期也能起到一定的保护作用。这一结果证明,可以通过在减毒鳗弧菌中利用表面展示将外源保护性抗原展示到细胞表面的方法开发多价疫苗。同时我们还将MCP融合到大肠杆菌α-溶血素分泌系统中,在大肠杆菌和鳗弧菌MVAV6203中考察其分泌情况。在大肠杆菌宿主中,可以检测到约400μg／L的MCP被分泌出来,然而鳗弧菌中却没有分泌。为了优化鳗弧菌疫苗株,以开发不含抗性标记的重组减毒鳗弧菌疫苗,我们尝试通过缺失relA、天冬氨酸半醛脱氢酶基因asd并插入TTaraC PBAD c2的方法在鳗弧菌中构建平衡-致死宿主载体系统。我们发现鳗弧菌中含有两个具有功能的asd基因asdA和asdB,且asdA和asdB都可以互补来源于其他菌种的asd突变株,但是asdA和asdB双突变对鳗弧菌却不致死。对AsdA和AsdB的生物学特性进行研究发现,AsdA与AsdB的3D结构有所不同,AsdB的结构更趋向于同一些革兰氏阳性菌的ASD结构相似,两个ASD的序列分析也显示了相同的结果。同时对鳗弧菌三磷酸甘油醛脱氢酶GAPDH的分析发现,该蛋白是被分泌到胞外的。更多还原

【Abstract】 Vibrio anguillarum is an important bacterial fish pathogen, responsible for both marine and freshwater fish epizootics throughout the world. In our previous work, several attenuated V. anguillarum strains derived from wild-type V. anguillarum strain MVM425 were constructed with recombinant DNA technology and proved to be excellent live vaccine candidates against Vibrio pathogens.For developing potential multivalent recombinant vaccines based on attenuated V. anguillarum live vaccine, efficient surface display systems are needed to display protective antigens onto the surface of live carrier. In this work, a series of novel cell surface display systems were examined by employing V. anguillarum outer membrane protein as anchoring motifs. These display systems consist of two parts. The first is the signal sequence and first eleven N-terminal amino acids of V. anguillarum outer membrane lipoprotein Wza, or the signal sequence and first nine N-terminal amino acids of the mature major Escherichia coli lipoprotein Lpp, and the second part is the transmembrane domains of V. anguillarum outer membrane proteins Omporfl, OmpU or Omp26La. Green fluorescent protein (GFP) was inserted to the systems and the results of GFP surface localization confirmed that four of the six surface display systems could successfully display GFP on Escherichia coli surface. The major capsid protein (MCP) of large yellow croaker iridovirus (LYCIV) was fused with Wza-Omporfl system and was displayed on the surface of an attenuated V. anguillarum MVAV6203, and a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of MVAV6203 to get multivalent vaccine candidates. The immune protection evaluation in zebra fish (Danio rerio) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger a full protection against V. anguillarum infection and an early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine.At the same time, the secretion of MCP based on the E. coliα-haemolysin transport system was investigated in both E. coli and attenuated V. anguillarum MVAV6203. With the aid of E. coli a-haemolysin transport system, about 400μg/L MCP was secreted into culture supernatant in E. coli, while intracellularly expressed MCP in MVAV6203 was not secreted. In order to optimize the V. anguillarum vaccine strain and develop an antibiotic-sensitive recombinant attenuated Vibrio vaccine, we try to develop a balanced-lethal host-vector system by delete the V. anguillarum relA gene, aspartate-semialdehyde dehydrogenase gene asd and insert TTaraC PBAD c2. We found that V. anguillarum contained two asd genes, asdA and asdB. Each of two genes functioned to complement different asd mutants from other bacterial species, and double deletions of asdA and asdB in V. anguillarum were not lethal. In the 3D structure analysis of AsdA and AsdB it was shown that AsdB isoform is quite different from the AsdA isoform and in fact more closely resembles the Gram-positive spASD structure. The result was confirmed in their alignment analysis. We also found that in V. anguillarum the glyceraldehyde-3-phosphate dehydrogenase can be secreted to the supernatant.更多还原