【作者】 孙云；

【导师】 孙黎；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2013， 博士

【摘要】 细菌性疾病是水产养殖业的重要疾病。迟缓爱德华氏菌（Edwardsiella tarda）是一种革兰氏阴性菌，能够感染多种经济鱼类并引起爱德华氏菌病，给水产养殖业造成巨大的经济损失。本论文主要是以牙鲆为实验动物，开展高效迟缓爱德华氏菌疫苗的设计与构建研究，并且探讨相关疫苗的免疫机制。具体如下：首先通过理论推断与实际实验相结合，本研究筛选出一系列迟缓爱德华氏菌免疫保护原，包括Esa1、Eta2、Eta1等，并在此基础上构建了基于细菌传递的重组亚单位疫苗、DNA疫苗、减毒疫苗和交叉保护疫苗。（1）基于Esa1构建重组亚单位疫苗、细菌传递的亚单位疫苗和DNA疫苗：Esa1是一个含有795个氨基酸残基的迟缓爱德华氏菌表面抗原，含有多个细菌表面抗原的保守位点且定位于迟缓爱德华氏菌的外膜。我们将Esa1在大肠杆菌中表达，获得了重组亚单位疫苗，发现其对迟缓爱德华氏菌引起的牙鲆感染有82%的免疫保护效应。随后我们利用肠道共生菌FP3构建了表面展示Esa1的细菌传递疫苗FP3/pJsa1。发现FP3/pJsa1作为口服微粒疫苗和注射疫苗分别能达到52%和79%的相对免疫保护率。我们进一步以Esa1为抗原，构建成了DNA疫苗pCEsa1。pCEsa1免疫牙鲆1个月和2个月后，其免疫保护率分别达到75%和71%。这些结果表明Esa1作为重组亚单位疫苗、鱼类肠道共生菌传递的表面展示型亚单位疫苗以及DNA疫苗皆具有良好的保护效应。（2）基于Eta2构建重组亚单位疫苗和DNA疫苗：在鱼类疫苗研究中，疫苗的免疫机制以及不同类型疫苗的免疫比较研究十分缺乏。本研究筛选得到一个新的迟缓爱德华氏菌免疫保护原Eta2，研究了Eta2作为重组亚单位疫苗和DNA疫苗所诱发的免疫保护效应及免疫机制。我们发现Eta2作为重组亚单位疫苗在免疫牙鲆4周和8周后，可分别产生83%和78%的相对保护率；作为DNA疫苗在免疫牙鲆4周和8周后产生的相对保护率分别为67%和68%。免疫学研究表明此两类疫苗均可增强牙鲆头肾巨噬细胞的杀菌活性、引起特异性和非特异性免疫相关基因上调，以及刺激牙鲆产生特异性抗体，其中亚单位疫苗诱发产生的抗体水平较高。进一步研究表明，Eta2作为重组亚单位疫苗主要引起牙鲆的体液免疫，而作为DNA疫苗则同时引起了鱼体B细胞和T细胞的免疫应答。这些结果说明Eta2是良好的免疫保护原，可以作为重组亚单位疫苗和DNA疫苗激发宿主的免疫保护反应，但是不同形式疫苗的免疫机制存在差异。（3）毒力因子Eta1及其免疫保护作用：在本研究中，我们发现了一个与细菌黏附素有同源性的体内诱导抗原Eta1。eta1基因在迟缓爱德华氏菌未侵染宿主时，其表达依赖于细菌的生长周期，而当迟缓爱德华氏菌侵染宿主时，eta1的mRNA水平显著上升。将eta1缺失突变后，缺失菌株在细胞和组织水平的侵染能力显著下降，说明Eta1是迟缓爱德华氏菌侵染所需的一个毒力因子。我们构建了Etal的重组亚单位疫苗rEta1，发现其能够诱导牙鲆产生高达83.3%的免疫保护率。rEtal免疫牙鲆后能够诱导鱼类产生特异性抗体，这些抗体在迟缓爱德华氏菌感染后能与表达于细菌表面的Eta1相结合。（4）减毒疫苗的构建以及疫苗导入途径研究：本研究筛选得到一株迟缓爱德华氏菌的弱毒菌株TX5RM，利用该弱毒菌株作为减毒疫苗，探索了注射、口服、浸泡及口服浸泡叠加等不同导入方式对疫苗免疫保护效应的影响。结果表明，这四种免疫方式均产生了明显的免疫保护效应，其中以口服与浸泡叠加的方式效果最为显著，在免疫牙鲆5周和8周后的相对保护率分别达到了80.6%和69.4%。（5）口服/浸泡DNA疫苗及其交叉免疫保护效应：我们将上述TX5RM作为DNA疫苗的导入载体，构建了携带海豚链球菌DNA疫苗pCS10的迟缓爱德华氏菌减毒疫苗TX5RMS10。免疫牙鲆后，TX5RMS10携带的DNA疫苗质粒能够在鱼体中稳定存在并表达海豚链球菌抗原Sia10。TX5RMS10经口服和浸泡方式免疫牙鲆1个月和2个月后，用迟缓爱德华氏菌和海豚链球菌分别攻毒，发现免疫组具有高达69~83%的存活率。这些结果说明TX5RMS10是一个高效的候选疫苗，其具有TX5RM和pCS10的免疫原性，因此可以诱导针对迟缓爱德华氏菌和海豚链球菌的交叉免疫保护效应。另外，该疫苗能够通过模拟自然感染的方式导入鱼体，从而避免了常规DNA疫苗的注射导入，因此在应用上具有非常好的优势。综上所述，本论文研究建立了新型鱼类疫苗的构建技术和导入方法，获得了多种不同类型的高效疫苗，初步阐明了不同类型疫苗诱发的免疫反应机制。这些研究结果促进了迟缓爱德华氏菌疫苗研究的发展，为鱼类迟缓爱德华氏菌病的防控提供了新思路。更多还原

【Abstract】 Bacterial pathogens are one of the major causes of aquaculture diseases.Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent ofa systematic fsh disease called edwardsiellosis, which affects a wide range of marineand freshwater fsh. The disease caused is often fatal and can lead to heavy economiclosses.Based on this situation, we in this study have designed and constructed vaccinesagainst E. tarda and examined the protective effects of the vaccines in a Japaneseflounder (Paralichthys olivaceus) model.We have so far constructed several different types of vaccines, which includeinactivated whole cell vaccines, attenuated live vaccines, DNA vaccines, cross-genusvaccines and recombinant subunit vaccines delivered by a live bacterial host. Thesevaccines proved to produce high levels (relative percentage survival greater than70%)of protection against E. tarda. We have identfied several E. tarda antigens, Esa1,Eta2and Eta1.Esa1is a D15-like surface antigen, which, when used as a recombinant subunitvaccine and DNA vaccine, are able to induce protective immunity in Japanesefounder (Paralichthys olivaceus) against E. tarda challenge. Taking advantage of thesecretion capacity of Esa1and the natural gut-colonization ability of a fsh commensalstrain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Vaccinationanalyses showed that FP3/pJsa1as an intraperitoneal injection vaccine and an oralvaccine embedded in alginate microspheres produced relative percent survival rates of 79%and52%. We examined further the immunoprotective potential of Esa1as aDNA vaccine. The DNA vaccine plasmid pCEsa1was constructed. Hence, comparedto PBS-vaccinated fsh, pCEsa1-vaccinated fsh were signifcantly protected, with aRPS of75%and71%respectively, at one-and two-month post-vaccination (p.v.).Together these results indicate that Esa1is a protective immunogen and an effectiveoral vaccine when delivered by FP3/pJsa1as a surface-anchored antigen.Eta2, a new E. tarda vaccine candidate, we have analyzed in a comparativemanner the immune response induced by Eta2in two different formfes:d purirecombinant subunit vaccine and DNA vaccine. Recombinant Eta2(rEta2) purfiedfrom Escherichia coli was highly protective against E. tarda challenge in a Japanesefounder model and produced relative percent of survival rates of83%and78%,respectively, at4-and8-week post-vaccination (p.v.). Eta2as a DNA vaccine in theform of plasmid pCEta2also induced strong protective immunity at4-and8-week p.v.Immunological analysis indicated that (i) rEta2and pCEta2enhanced head kidneymacrophageactivation;(ii) rEta2and pCEta2induced similar patterns of serumantibody production;(iii) both rEta2and pCEta2upregulated the expression ofspecifc and nonspecifc immune factors which include. Taken together, these resultsindicate that both rEta2and pCEta2induce sfpce ciand nonspecifc immunities,however, pCEta2induces both B cell and T cell responses, whereas rEta2inducesmainly humoral response.Eta1, we identifed, via in vivo-induced antigen technology. eta1expression wasgrowth phase dependent and reached maximum at mid-logarithmic phase. Duringinfection offounder lymphocytes, eta1expression was drastically increased at theearly stage of infection. Compared to the wild type, the eta1-defective mutant, TXeta1,was unaffected in growth but exhibited attenuated overall virulence, reduced tissuedissemination and colonization capacity, and impaired ability to infvoaduenderlymphocytes and to block the immune response of host cells. Furthermore, when usedas a subunit vaccine, rEta1produced relative percent of survival rates of83.3%infounder against lethal E. tarda challenge. Taken together, these results indicate thatEta1is an in vivo-induced antigen that mediates pathogen-host nteraction and, as a result, is required for optimal bacterial infection.In this study, we also have selected TX5RM, which is an attenuated E. tardastrain with good vaccine potential and that a combination of oral and immersionvaccinations may be a good choice for the administration of live attenuated vaccines.The results showed that TX5RM administered via all four approaches producedsignifcant protection, with the highest protection levels observed with TX5RMadministered via oral feeding plus immersion, which were, in terms of relative percentof survival (RPS),80.6%and69.4%at5-and8-week post-vaccination, respectively.We have constructed a cross-genus vaccine by takeing advantage of theresidual infectivity of TX5RM and using it as a carrier host for the natural delivery ofa S. iniae DNA vaccine. The recombinant TX5RM, TX5RMS10, was created, whichharbours and retains stably the DNA vaccine plasmid pCS10that expresses Sia10.Following E. tarda and S. iniae challenge at one and2months p.v., the vaccinatedfsh exhibited relative percent survival rates of69–83%. Immunological analysisindicated that TX5RMS10-vaccinated fsh produced specifc serum antibodies andexhibited enhanced expression of a wide range of immune genes.In addition, we also examined the immune response of the vaccines and foundthat DNA vaccines stimulate stronger systematic immunity than subunit vaccines.These findings promote the research on the marine fish vaccines and provide insightsto the protection mechanism of vaccines.更多还原