Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain

The gene encoding the Edwardsiella tarda ferric uptake regulator (Fur(Et)) was cloned from a pathogenic E. tarda strain isolated from diseased fish. Fur(Et) shares 90% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and was able to complement the mutant phenotype of a fur(Ec)-defective E. coli strain. Mutational analysis indicated that C92S and C95S mutations inactivated Fur(Et) whereas E112K mutation resulted in a superactive Fur(Et) variant. Fur(Et) negatively regulated its own expression; interruption of this regulation impaired bacterial growth, altered the production of certain outer membrane proteins, and attenuated bacterial virulence.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

迟缓爱德华氏菌(Edwardsiella tarda) LuxS/AI-2群体感应系统研究

从患病牙鲆中分离出迟缓爱德华氏菌株TX1，经报告菌株检测发现TX1有AI-2活性。用梯度PCR和Genome walking的方法克隆了TX1 luxS基因，将luxS基因在大肠杆菌DH5α中表达，证明其具有功能活性。在TX1中，luxS的表达与AI-2的活性基本是一致的，二者均受生长时期和生长条件的调节，即在glucose存在的条件下luxS表达和AI-2活性升高，而在高温条件下luxS表达和AI-2活性降低。glucose对AI-2活性以及luxS表达的影响经过荧光定量PCR，启动子活性检测，AI-2活性检测以及凝胶滞缓等一系列的实验证实是由cAMP-CRP复合物介导的，该复合物可以通过与luxS启动子相互作用而抑制luxS的表达。RNA干扰表明，TX1中luxS表达被干扰以后，对细菌产生了多方面的影响，包括：(1) 降低AI-2水平；(2) 降低细菌的生长能力；(3) 降低Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成能力；(4) 减弱细菌毒力。外源AI-2的添加可以回复Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成，但是并不能修复生长状况，表明LuxS在TX1中具有双重功能，即参与细胞代谢以及群体感应信号传导。基于LuxS/AI-2群体感应系统对细菌毒力的重要性，设计并筛选了一个该系统的阻遏因子5411。Pull-down实验证明5411可以和LuxS特异性结合。研究表明5411在TX1中表达导致细菌毒力显著下降。将5411克隆至牙鲆共生菌FP3中，发现5411可以被分泌到胞外并能被TX1吸收。将表达5411的共生菌导入牙鲆，发现其能够有效阻遏TX1对牙鲆的侵染。 这些结果表明：(1) TX1中AI-2的活性受控于LuxS，而后者则受生长时期和生长条件的调控；(2) luxS的正常表达对于细菌的正常生长和侵染是必需的；(3) LuxS/AI-2群体感应系统调控Ⅲ型分泌系统相关毒力因子的表达；(4) 通过阻遏LuxS/AI-2群体感应系统来抑制病原菌侵染是一种具有潜力的新型病害防控方法。

Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.

Immunoprotective analysis of VhhP2, a Vibrio harveyi vaccine candidate

VhhP2 is an Outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a Subunit Vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) II alpha. A VhhP2 surface display system, in the form of the fish commensal strain FIR harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited Strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the Surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5 alpha/pJVP18, which expresses and surface-displays the VhhP2-Et18 chimera, proved to be an effective vaccine that call protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as F. tarda. (C) 2009 Elsevier Ltd. All rights reserved.

Differential regulation of Sciaenops ocellatus viperin expression by intracellular and extracellular bacterial pathogens

Viperin is an antiviral protein that has been found to exist in diverse vertebrate organisms and is involved in innate immunity against the infection of a wide range of viruses. However, it is largely unclear as to the potential role played by viperin in bacterial infection. In this study, we identified the red drum Sciaenops ocellatus viperin gene (SoVip) and analyzed its expression in relation to bacterial challenge. The complete gene of SoVip is 2570 bp in length and contains six exons and five introns. The open reading frame of SoVip is 1065 bp, which is flanked by a 5'-untranslated region (UTR) of 34 bp and a 3'-UTR of 350 bp. The deduced amino acid sequence of SoVip shares extensive identities with the viperins of several fish species and possesses the conserved domain of the radical S-adenosylmethionine superfamily proteins. Expressional analysis showed that constitutive expression of SoVip was relatively high in blood, muscle, brain, spleen, and liver, and low in kidney, gill, and heart. Experimental challenges with poly(I:C) and bacterial pathogens indicated that SoVip expression in liver was significantly upregulated by poly(I:C) and the fish pathogen Edwardsiella tarda but down-regulated by the fish pathogens Listonella anguillarum and Streptococcus iniae. Similar differential induction patterns were also observed at cellular level with primary hepatocytes challenged with E. tarda, L anguillarum, and S. iniae. Infection study showed that all three bacterial pathogens could attach to cultured primary hepatocytes but only E. tarda was able to invade into and survive in hepatocytes. Together these results indicate that SoVip is involved in host immune response during bacterial infection and is differentially regulated at transcription level by different bacterial pathogens. (C) 2010 Elsevier Ltd. All rights reserved.

牙鲆和大黄鱼经济性状候选基因的DNA分子标记研究

本文利用不同的分子标记方法，分别对牙鲆及大黄鱼不同养殖群体的生长、抗病等经济性状的候选基因进行了序列多态性研究，检测到了几个SNP位点和微卫星的多态性位点，并分析了它们与经济性状之间的相关性；同时，利用微卫星的多态性位点对牙鲆2个养殖群体的遗传变异进行了分析，这些均为海水鱼类遗传育种及标记辅助选育工作提供了基础数据。 在牙鲆胶南养殖群体中，以100个个体为实验材料，根据其生长激素（GH）基因的6个外显子序列设计引物，通过SSCP分析技术显示该群体GH基因的第4外显子存在多态性，检测到2种基因型，AA型和AB型。DNA测序结果表明，AB型在第1763位发生碱基突变，c→t，与AA型同源性达到99%。连锁分析结果表明：这2种基因型的个体在体重和头长上表现出显著的差异，AB型个体的体重和头长都明显大于AA型个体(P<0.05)，由此推测等位基因B是一个对牙鲆体重和头长都有利的等位基因；这2种基因型个体之间在其体型性状上也存在显著差异（P<0.05）；同时，该多态位点的Hardy-Weinberg平衡性检验结果表明，该群体处于Hardy-Weinberg平衡状态。在牙鲆GH基因第1外显子区域还发现了一个微卫星位点，对该位点进行多态性分析，检测到5种基因型、3种等位基因，one-way ANOVA统计结果显示，基因型AC个体的体重、头长和体高明显大于其它基因型个体（P<0.05），C是一个对体重、头长和体高有利的等位基因。 对2个大黄鱼养殖群体的GH基因进行SSCP分析后发现，浙江群体大黄鱼GH基因在第196位存在1个SNP（g→a）位点，检测到2种基因型，AA和AB。t检验结果表明，AA型个体的体高比AB型个体的高(P≤0.05)，但AB型个体在体长/体高上占优势(P≤0.05)，提示该突变位点可以作为大黄鱼体型性状的候选标记。福建群体大黄鱼GH基因在第692位有1个SNP位点(t→c)，共检测到2种基因型，CC型和CD型，其中，CD基因型个体的体重和全长显著大于CC基因型个体(P≤0.01)，提示该位点可以作为大黄鱼体重和头长性状的候选标记。 在牙鲆胶南和日照2个养殖群体中，采用牙鲆GHR基因5’端Promoter区的一个微卫星标记，进行了群体遗传变异的研究，并探索了该基因多态性位点与牙鲆生长性状之间的相关性。结果表明，2个群体在该位点检测到的等位基因数为12和9个，有效等位基因数为6.26和5.04个。两个群体该位点的Hardy-Weinberg遗传偏离指数均为正值，并没有显示出杂合子缺失，但各基因型分布频率都在一定程度上偏离Hardy-Weinberg平衡(P<0.01)。连锁分析发现，在胶南群体中，IM基因型对应的个体在全重、全长、体长、头长、体高和眼径形态学数据中均是最大的，但仅在体重上极其显著的大于全部其它基因型个体；在日照群体中，BC基因型对应的个体在全重、全长、体高、尾柄高、尾柄长和眼径数据中均是最大的；而CJ基因型对应的个体在体长和头长这两组数据中是最大的。由此认为，该位点IM基因型可以作为牙鲆体重性状的潜在标记。 在进行牙鲆抗病性状标记的筛选时，利用迟缓爱德华氏菌（Edwardsiella tarda）LSE40对牙鲆鱼进行攻毒感染实验，得到死亡群体和未死亡群体。选择Toll样受体基因中的TLR2、TLR3和TLR9基因作为候选基因，分别对这3个基因中的部分序列共设计7对引物进行扩增，同时对扩增产物进行RFLP多态性分析，目前只在TLR3基因内检测到一个EcoRI的酶切多态性位点，测序后发现，这是由于在TLR3基因第3806位的EcoRI酶切位点在某些个体中缺失所致。酶切产物共呈现出3种基因型，分别定义为AA，AB和BB。χ2检验证明该多态性位点与牙鲆抗迟缓爱德华氏菌LSE40的能力有一定关系。利用多因素非条件Logistic回归分析对死亡组和存活组牙鲆的各种形态学数据以及不同基因型之间进行了分析，发现体长、头长和体高均具有显著的相关性（P<0.05），而这几个因素与体重的相关性不显著（P>0.05）。多因素非条件Logistic分析后发现：AA基因型对死亡率具有显著的影响（P<0.05），是主要的危险因素，而AB基因型的作用不显著（P>0.05）；头长是主要的保护因素（P<0.05），体重对死亡率的影响很小。χ2检验证明，等位基因A是对死亡的主要危险等位基因，B是对存活有利的主要等位基因。推测该位点可以作为牙鲆抗迟缓爱德华氏菌的潜在标记。

迟缓爱德华氏菌III型分泌系统(T3SS)输送器蛋白及分子伴侣的研究

迟缓爱德华氏菌（Edwardsiella tarda）是重要的革兰氏阴性致病菌，可以感染包括人类在内的多种动物。由迟缓爱德华氏菌引发的爱德华氏菌病已经在许多水产养殖动物中被发现，每年给淡水和海水水产养殖业带来巨大的损失。目前为止对于迟缓爱德华氏菌病的防治以化学治疗为主，疫苗的研究还在进行中。III型分泌系统（T3SS）是E. tarda重要的致病因子，虽然T3SS基因簇的结构及部分基因的功能得到了初步研究，但其作用机制还未得到阐明。本研究着重开展了迟缓爱德华氏菌T3SS输送器蛋白EseC的分子伴侣的鉴定及功能研究，并对输送器蛋白及其免疫功能进行了初步探讨，希望进一步地了解T3SS在E. tarda致病中的功能及其在疫苗研制中的作用。 一、迟缓爱德华氏菌III型分泌系统（T3SS）输送器蛋白EseC分子伴侣的鉴定和功能研究 以前的工作表明，EseB、EseC和EseD蛋白是E. tarda T3SS输送器蛋白的组成成分，在分泌到细菌细胞外后可以组成输送器装置。分子伴侣对于输送器蛋白的稳定和分泌具有重要的作用，EscC已经被鉴定为EseB和EseD的分子伴侣，而EseC的分子伴侣还没有得到鉴定。在本实验中，我们以EseC作为研究对象，主要开展了EseC分子伴侣鉴定的研究。 生物信息学分析表明，在E. tarda T3SS基因簇上的escA基因与eseC相邻，其编码的蛋白形成一个大的螺旋结构，为分子量较小（17.5kD）的酸性蛋白（pI 4.79），并与已鉴定的分子伴侣具有序列的同源性，这些符合细菌T3SS分子伴侣的特征。研究发现，EscA蛋白分布在细菌的细胞质和细胞膜上。在escA基因缺失后，大大降低了EseC分泌到细菌细胞外的量，同时EseC蛋白在细菌细胞质中的积聚量也减少，当escA基因缺失突变株得到escA基因互补后，EseC的分泌和在细胞质内的积聚恢复到了野生型菌株水平。氯霉素阻断蛋白质合成的实验发现，当细菌不表达EscA的情况下，EseC蛋白逐渐降解，说明了EscA可以影响EseC在胞质中的稳定。蛋白体外结合试验和免疫共沉淀实验发现，EseC和EscA在体外可以结合，在细菌细胞质中也可以相互结合，表明EseC和EscA可以相互作用。上述结果表明，EscA是EseC的分子伴侣。 在确定了EscA是EseC的分子伴侣之后，我们进一步确定EscA对EseC表达的影响，以及两者相互作用的结构域。通过检测转录水平和翻译水平的EseC-LacZ融合蛋白表达情况，发现在EscA缺失的情况下，EseC的转录水平没有变化，而翻译水平下降，表明EscA对EseC的影响在转录后水平。通过构建含有部分结构域缺失的escA或eseC的体外共表达体系，并进行Western blot分析，确定了EseC的31-137氨基酸序列为与EscA结合的区域，而在EscA中并没有找到与EseC结合的区域。EseC的31-137氨基酸片段缺失后，EseC的分泌和在E. tarda细菌细胞中的积聚下降，其下降幅度与escA突变株相当，进一步表明EseC的31-137氨基酸为与EscA相互作用的区域。最后人工感染实验表明，分子伴侣EscA及其与EseC的相互作用对E. tarda的致病力有影响。 二、迟缓爱德华氏菌T3SS输送器蛋白的研究 一些研究表明，T3SS在细菌与宿主相互作用的过程中表达，在体外诱导的条件下也可表达。为了确定E. tarda T3SS体外诱导表达的条件，我们检测了不同培养温度、pH条件下，E. tarda T3SS输送器蛋白表达的情况。研究表明，37°C条件下，E. tarda生长快，T3SS的输送器蛋白表达较低；28°C条件下，T3SS的输送器蛋白表达最高，而在20°C条件下，没有检测到T3SS输送器蛋白的表达。在28°C和37°C的培养条件下，中性和碱性相对酸性来说适合细菌的生长和T3SS输送器蛋白的表达。我们分析了E. tarda野生型和输送器蛋白突变株中的输送器蛋白的细胞分布，并据此推测输送器形成的机制。单一输送器蛋白的缺失不影响其它两个输送器蛋白的积聚，而输送器蛋白的分泌之间存在一定的相互影响。 通过检测输送器蛋白突变株ΔeseB, ΔeseC, ΔeseD生长、泳动、自凝聚和溶血能力的变化，发现在输送器蛋白基因缺失后，体外培养的E. tarda的生长速度变慢，泳动、自凝聚和溶血能力也变弱，说明了输送器蛋白在细菌的生长和功能行使中的重要作用。 为了检测输送器蛋白的免疫保护效果，我们克隆了eseD基因，将其在表达菌株BL21(DE3)中进行表达，并将重组表达的EseD蛋白经Ni-NTA树脂进行纯化。以EseD纯化蛋白作为蛋白抗原对大菱鲆进行注射，EseD蛋白表现出了对鱼类的免疫原性，其抗体效价在第7周达到了最高，为1：5120。攻毒实验表明该蛋白对于保护大菱鲆免疫E. tarda的感染具有帮助作用，在105cfu攻毒浓度下大菱鲆的相对存活率(RPS)为62.5%。结果说明EseD蛋白可以作为蛋白抗原疫苗的候选，并能够在保护鱼类免疫爱德华氏菌病中发挥作用。

鱼类疫苗的研究：I: 海水鱼类病原菌乳化口服疫苗的研究；II：迟缓爱德华氏菌和嗜水气单胞菌外膜蛋白疫苗的研究

本论文的目的是研究几种病原菌口服疫苗接种鱼类的免疫效果，并从常见病原菌株中筛选几个具有较好保护效果的蛋白抗原，利用口服免疫的方式，接种养殖动物，并检测免疫效果。 以10号白油为有机溶剂，采用搅拌与均浆方法制备鳗弧菌M3和SMP1的油乳化二价疫苗，用饵料包埋后以口服途径免疫养殖大菱鲆，评价免疫大菱鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别连续口服免疫大菱鲆一周后，在后肠组织，乳化疫苗刺激产生的非特异性活力、特异性抗体水平均高于未乳化疫苗；而在血清，两种疫苗引起的两种酶的活力、SMP1抗体水平没有变化，但在乳化疫苗免疫的大菱鲆检测到明显高于未免疫对照大菱鲆的M3抗体水平。大菱鲆后肠组织原位杂交结果显示，口服免疫的大菱鲆后肠褶皱有IgM抗体的产生和分布。其中，乳化疫苗免疫大菱鲆的IgM抗体的产生和分布水平高于未乳化疫苗免疫的大菱鲆。攻毒实验显示，乳化疫苗免疫的大菱鲆对M3和SMP1的感染分别获得100%和50%的免疫保护率，而未乳化疫苗获得的免疫保护率分别为57.9%和0%，表明乳化疫苗比未乳化疫苗更有效地保护大菱鲆、抵抗病原的感染。在乳化疫苗免疫持续期的研究中，免疫的大菱鲆后肠在免疫后120天仍能检测到抗体效价，在免疫后90天还能观察到一定的免疫保护效果。免疫30天、60天、90天和120天的大菱鲆分别获得100%、66.7%、36.7%和13.3%的免疫保护力。 以鳗弧菌M3和SMP1、链球菌CF、迟缓爱德华菌SMW7作为细菌抗原制备油乳化多价口服疫苗和轮虫携带疫苗，口服途径免疫养殖大菱鲆与大菱鲆初孵仔鱼。结果显示，在免疫大菱鲆后肠可检测到抗M3抗体水平的提高（P<0.05），而在其胆汁、鳃、中肠、体表黏液、前肠与血清中抗体效价变化与对照组没有显示出差异；没有检测到免疫大菱鲆后肠抗SMP1、SMW7、CF抗体效价。M3浸泡攻毒实验显示，口服免疫的大菱鲆获得了100%的免疫保护力；在M3注射攻毒和SMP1、CF、SMW7浸泡攻毒大菱鲆的实验中，在每个攻毒组中，免疫组大菱鲆开始死亡的时间都要比对照组有不同程度的延迟，但攻毒大菱鲆都发生死亡，不能显示出与对照组的差异。轮虫携带免疫的结果显示，免疫的大菱鲆初孵仔鱼并未获得较好的保护效果，与对照大菱鲆没有体现出差异。 从致病性病嗜水气单胞菌（Aeromonas hydrophila）LSA34克隆并表达ahaI基因和gapA基因，从迟缓爱德华菌（Edwardsiella tarda）LSE40克隆并表达eseB，将所得蛋白分别通过腹腔注射途径免疫大菱鲆，检测蛋白的免疫原性和免疫保护。结果在免疫后7天就可以检测到AhaI、GapA蛋白免疫组大菱鲆产生的抗体，至第40天可以检测到明显的保护性抗体，之后抗体效价增加明显，直至第60天时达到最高值。EseB免疫的大菱鲆第一次免疫后15天就有较高的抗体效价产生，明显高于对照组大菱鲆血清抗体效价，到距第一次免疫60天时，抗体效价达到最高值。攻毒实验显示，与对照组相比，AhaI免疫组和GapA免疫组对LSA34感染的免疫保护力分别为80%和100%；AhaI免疫组和GapA免疫组对LSE40感染的免疫保护力分别为30%和10%。，而对照组牙鲆对人工攻毒不具有保护力。以AhaI和GapA作为疫苗免疫大菱鲆，使大菱鲆获得了对嗜水气单胞菌LSA34较高的免疫保护；而对迟缓爱德华氏菌LSE40的交叉保护能力没有明显提高。EseB免疫的大菱鲆在攻毒实验中并没有显示出较好的保护效果，与对照组相比，只是在死亡时间上有所延迟。 以从致病性嗜水气单胞菌中克隆的ahaI和gapA基因表达出的蛋白为蛋白抗原制备油乳化疫苗，用饵料包埋后以口服途径免疫养殖牙鲆，评价免疫牙鲆的免疫应答和疫苗的保护效果。结果显示，以油乳化和未油乳化疫苗分别免疫牙鲆一周后，在后肠组织，AhaI和GapA乳化疫苗免疫组牙鲆检测到抗体，且分别高于AhaI和GapA未乳化疫苗免疫的牙鲆；而在血清，GapA的两种疫苗引起的GapA抗体水平没有变化；但在AhaI乳化疫苗免疫的牙鲆第21天和第35天的血清中检测到高于未免疫对照牙鲆的AhaI抗体水平，AhaI未乳化疫苗免疫牙鲆血清对照组相比没有检测到AhaI抗体水平的变化。

迟缓爱德华氏菌两种保护性抗原及AcrAB耐药系统的分析

迟缓爱德华氏菌是危害水产养殖业发展的重要病原菌之一，因而其免疫防治研究具有重要意义。论文分析了9种具有保护潜能的迟缓爱德华氏菌蛋白，经过牙鲆免疫保护实验，筛选出EseD和Et18两种有显著性保护效应的抗原。为了提高其保护效应，论文使用基因工程技术将这两种抗原融合到一起，构建重组融合蛋白EEH。结果表明，融合蛋白EEH保护效应较EseD和Et18分别免疫时有所提高。ELISA和Western blotting 结果显示，三种蛋白都能诱导牙鲆产生特异抗体。这些研究为开发迟缓爱德华氏菌疫苗提供了理论基础。 论文克隆分析了迟缓爱德华氏菌AcrAB耐药系统，采用定点突变确定了acrAB、acrR的启动子序列和AcrR在acrAB启动子的结合位点。启动子分析显示，AcrR对acrAB启动子有300倍抑制效应, 对acrR启动子有3倍抑制效应。定点突变显示，K39和R45对AcrR功能具重要性；缺失突变表明，N端205个氨基酸残基是其功能必需。实验筛选出Acriflavine、Ethidium Bromide、Methyl Viologen、Sodium Dodecyl Sulfate等四种AcrR诱导物。分析AcrR过量表达菌株结果显示，其耐药性、生长状况和毒力水平较阴性对照组降低。这些研究加深了我们对迟缓爱德华氏菌耐药机制及其与毒力关系的了解。

迟缓爱德华氏菌基因组fosmid文库的构建、opp基因簇的克隆及aroA基因缺失突变株的构建

本研究构建了迟缓爱德华氏菌(Edwardsiella tarda)LSE40 基因组fosmid文库，该文库共包含2 500个克隆，插入片段平均大小为33.6kb，文库总容量约84Mb，覆盖E.tarda LSE40基因组（按5Mb计算）超过16倍。随机挑取1 000个fosmid克隆进行双末端测序共得到1 741条高质量的序列，序列平均长度546 bp，全长949 997bp，约为E.tarda基因组的19％。将这些序列提交到KEGG自动注释服务器KAAS对所得序列进行代谢途径分析，得到E.tarda LSE40的KO (KEGG Orthology) 注释。分析结果表明，与代谢途径相关的基因有932条序列，与环境信息处理相关基因283条，与遗传信息处理相关基因220条，与细胞进程和人类疾病相关的基因分别为64条和16条。同时将序列进行BlastX，按照微生物致病性共同主题找到61个毒力相关基因。Fosmid文库的建立和部分基因组序列的生物信息学分析为进一步研究E.tarda LSE40的致病机制、代谢机制和生理生态机制提供了丰富的物质基础。 通过比较基因组的方法，从E.tarda LSE40 fosmid文库克隆到编码寡肽透过酶的opp基因簇，该基因簇全长6 741bp，含有5个ORF，依次编码OppA-B-C-D-F 5个蛋白；位于oppA和oppB的间隔区和oppF之后的非编码区各有一个茎环结构，推测分别为oppA和opp基因簇的转录终止子。以细菌OppA的保守结构域SBP_bac_5构建系统发生树，结果显示E.tarda LSE40与同属细菌E.ictaluri的亲缘关系最近，与肠杆菌科细菌的亲缘关系较近，与革兰氏阳性细菌的亲缘关系较远，表明OppA的SBP_bac_5结构域可作为细菌分类鉴定的依据。 从E.tarda LSE40 fosmid文库克隆aroA基因全序列，该序列全长1 287bp，编码428个氨基酸，与鲶鱼爱德华氏菌(E. ictaluri)氨基酸相似性在94%，与其他肠杆菌科菌如Escherichia coli和Yersinia enterocolitica相似性在73%-74%。通过In-frame deletion构建了E.tarda LSE40 aroA缺失突变株。与野生型相比，aroA突变株的半数致死量LD50提高了62倍。在牙鲆接种~106cfu/ml的E.tarda细菌时，接种野生型细菌的牙鲆在6天内全部死亡，濒死鱼的细菌数达7.97×108cfu/ 100mg；而接种aroA突变株的牙鲆没有出现死亡，28天后检测不到细菌的存在。实验结果为进一步评价aroA突变株作为减毒活疫苗打下了基础。

大鸨生长期能量代谢和蛋白质沉积量的初步研究

对不同日龄笼养大鸨(Otis tarda)的能量代谢和蛋白质沉积量进行研究.结果表明,50日龄、70日龄、300日龄和400日龄雄性大鸨的能量日摄入量分别为1 498.9 kJ、2 376.2 kJ、2 397.4 kJ和2 465.9 kJ;能量代谢率分别为82.3%、81.8%、81.8%和83.1%;蛋白质的日沉积量分别为5.8 g、13.4 g、15.8 g和13.5 g. 表3 参9