【作者】 牟为；

【导师】 张元兴； 刘琴；

【作者基本信息】 华东理工大学 ， 海洋生物学， 2011， 硕士

【摘要】 重组细菌载体疫苗作为一种有效的策略可以携带抗原蛋白并激发宿主的产生多重免疫保护力。目前,利用细菌载体活疫苗作为递呈载体是新兴现代疫苗的重要方向,接种这种疫苗后,机体既可产生针对载体细菌来源的病原的免疫保护力,又可产生针对携带抗原来源病原的免疫保护力,能够达到同时预防多种病原感染的作用。然而,活菌载体的安全性、外源基因表达稳定性等问题也是细菌载体疫苗发展中不可忽视的问题。迟钝爱德华氏菌(Edwardsiella tarda)是一种重要的革兰氏阴性鱼类病原菌。该病原菌能引起鱼类的出血性败血症,在本实验室前期工作中,已经从发病大菱鲆体内分离得到一株迟钝爱德华氏菌EIB202强毒株,并对其进行了全基因组测序,并构建了一系列适用于载体活菌疫苗的缺失减毒菌株。在本研究中,我们筛选了53个不同细菌来源的铁调控启动子,并检测了它们在体外培养时的转录活性。将筛选得到的具有较好铁调控活性的启动子Pdps和Pdps在巨噬细胞内进行胞内转录活性分析,最终选取了能够在胞内高效表达的启动子Pdps,并在模式动物斑马鱼上进行了观察检测。实验证明Pdps在体内有一个转录增强的趋势,可以用于体内诱导表达系统的构建。为了证明该系统能够作为成熟的抗原表达系统,我们将来源于嗜水气单胞菌(Aeromonas hydrophila) LSA34中的保护性抗原GAPDH连入含有Pdps的重组质粒中,同时,选取迟钝爱德华氏菌的减毒株WED作为宿主菌,构建了细菌载体疫苗WED/pUTDgap.大菱鲆免疫攻毒实验证明,WED/pUTDgap针对对野生型迟钝爱德华氏菌和嗜水气单胞菌均有超过60%的免疫保护力。本研究在迟钝爱德华氏菌中成功建立了基于铁诱导启动子的体内诱导抗原表达系统,并在迟钝爱德华氏菌减毒株WED中实现了嗜水气单胞菌保护性抗原蛋白的体内表达,成功构建了一株具有二价免疫保护力的迟钝爱德华氏菌减毒活疫苗候选株。更多还原

【Abstract】 Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of bacterial vector vaccine is to establish a stable antigen expression system in vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are good bacterial vectors for use in recombinant vaccine design. In this study, we aimed to build an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda vector vaccine. With wild-type strain E. tarda EIB202 as a vector,53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro, and the promoters Pdps and PyncE showed high transcription activity. These two promoters were further assayed for their transcription profile in vivo, and Pdps demonstrated an enhanced in vivo-inducible transcription in macrophage, zebrafish larvae and zebrafish adults. The gapA34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the Pdps-based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant vector vaccine WED/pUTDgap was evaluated in turbot(Scophtalmus maximus). Over 60%of the vaccinated fish survived when challenging with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the Pdps-based antigen delivery system had great potential in bacterial vector vaccine application.更多还原