【作者】 王雪鹏；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 水生生物学， 2009， 博士

【摘要】 迟缓爱德华菌(Edwardsiella tarda)是危害水产养殖的病原菌之一,可引起多种类型的感染,致病机理复杂。由迟缓爱德华菌引起的疾病在世界海水、淡水养殖动物中普遍流行。化学药物是治疗迟缓爱德华菌感染的一种重要手段,但又会引起食品药物残留、细菌耐药性等问题。尽管预防迟缓爱德华菌感染的疫苗报道很多,但这些常用疫苗多是福尔马林(或热)灭活疫苗或亚单位疫苗,影响了细菌表面抗原的物理化学结构,影响了疫苗的免疫效果,而一些亚单位疫苗又缺少足够的免疫原性,常需加入免疫佐剂使用,而这些免疫佐剂有可能具有副作用。细菌菌蜕是通过调控噬菌体PhiX174的裂解基因E在革兰氏阴性细菌中表达而形成的无细胞浆和繁殖能力的细菌空壳,因其保持了细菌原有的细胞形态、细菌表面抗原性和黏附性等特性,即使象菌毛一样脆弱的结构也能被保护。同时,菌蜕包含LPS、类脂、肽聚糖等天然的免疫刺激复合物,无须使用佐剂。因此,菌蜕疫苗能诱导更强的免疫反应,是一种比常规疫苗更理想的新型疫苗体系。本研究首次在国内报道迟缓爱德华菌菌蜕疫苗的制备及应用。1迟缓爱德华菌制苗菌株的筛选通过形态学观察、选择性培养基筛选、生理生化分析、PCR鉴定及16S rDNA序列测定,证明本实验室保存的细菌为迟缓爱德华菌。应用PCR方法检测了迟缓爱德华菌的6个毒力基因在国内外分离株中的分布情况,并根据其溶血活性、对斑马鱼和小鼠的致病性研究,筛选出强毒株,为进一步后续研究奠定了基础。2迟缓爱德华菌菌蜕疫苗的制备本研究成功构建溶菌质粒载体,并将其转入迟缓爱德华菌真鲷分离株,通过温度诱导并跟踪检测其溶菌动力学过程,制备出迟缓爱德华菌菌蜕疫苗。扫描电镜和透射电镜观察,证实细菌结构未改变,且不含内容物。3以小鼠动物模型评价迟缓爱德华菌菌蜕疫苗的口服免疫效果分别用菌蜕疫苗(ETG)、福尔马林全菌灭活疫苗(FKC)和PBS口服免疫小鼠,并跟踪检测其体液免疫水平和细胞免疫水平的变化。结果表明,ETG免疫组血清IgA和IgG的抗体滴度与FKC和PBS免疫组相比,差异显著；ETG免疫组小鼠外周血淋巴细胞中CD3+／CD4+／CD8+阳性的百分率与FKC和PBS免疫组相比,差异显著。同源菌攻击试验表明,ETG、FKC和PBS组的相对保护率分别为86.7%(26／30),73.3%(22／30)和33.3%(10／30)。结果表明,ETG疫苗能诱导强烈的细胞免疫和体液免疫,提高免疫保护能力。4迟缓爱德华菌菌蜕疫苗对斑马鱼的免疫保护试验详细阐述研究斑马鱼白细胞吞噬活性的方法,并研究了迟缓爱德华菌蜕疫苗对斑马鱼白细胞吞噬活性的影响。试验表明,ETG免疫组的白细胞吞噬活性明显高于FKC和PBS免疫组,差异显著。攻击试验结果显示,ETG的免疫保护率高达83.3%(25／30)与FKC和PBS免疫组的70%(21／30)和6.7%(2／30)相比,差异显著。结果表明,ETG疫苗能有效地激活鱼类的免疫系统,产生免疫保护。5应用裂解基因和核酸酶基因共表达制备菌蜕疫苗葡萄球菌核酸酶A(SNA)对单链、双链DNA或RNA均具有较强的降解能力。成功构建表达葡萄球菌核酸酶A不同片段的质粒载体,通过诱导表达发现酶活性只与N’末端氨基酸序列有关,并发现编码SNA N’末端26个氨基酸的序列与λ噬菌体Cro基因融合表达可以降解细菌核酸,又可以被入噬菌体的cI857ts／PR调节系统调控。因此,利用这一技术成功构建调控噬菌体裂解基因和葡萄球菌核酸酶基因先后表达的质粒载体,制备出安全性更高的菌蜕疫苗。更多还原

【Abstract】 Edwardsiella tarda (E. tarda) is widely distributed in aquatic environments and is infectious to variety of animals including humans, fish, amphibians, reptiles and birds. This organism’s versatility with respect to the broad-range of hosts highlight the importance of developing strategies for the protection of both animals and humans from E. tarda infections.In recent years, chemotherapy has been used effectively in controling fish infections, however, there is significant concern regarding food safety following chemotherapeutic interventions in addition to the danger of selecting for antibiotic-resistant E. tarda isolates which have been reported worldwide. These concerns have prompted the development of novel vaccination strategies for the control of E. tarda infections. Although the development of E. tarda vaccines has been attempted, their efficacy against challenge has been inconsistent. The commercial vaccines presently available consist of heat- or formalin-inactivated E. tarda or subunit formulations, however these strategies can affect the physio-chemical structural properties of surface antigens thereby negatively affecting the development of protective immunity. Subunit vaccines are often less immunogenic, necessitating the use of adjuvants which may have significant negative side effects on the host.Bacterial ghosts are empty cell envelopes that are produced, for example, by the controlled expression of the PhiX174 lysis gene E in Gram-negative bacteria. Expression of lysis gene E leads to the formation of trans-membrane tunnels which consequently lead to the loss of cytoplasmic contents. The resulting bacterial ghosts have been demonstrated to retain functional and antigenic determinants of the envelope. Even highly-sensitive and fragile structures such as pili are well protected following ghost formation. These data suggested that ghosts could be used in place of the traditional live-attenuated vaccine preparations to elicit immunity.In the present study, E. tarda ghosts were generated and used as vaccine candidates. 1 Screening of Edwardsiella tarda Strain for Ghost VaccineThe applicability of morphology, selective culture medium, biochemical tests, PCR and 16S rDNA methods for identification of E. tarda associated in fish and human culture system was studied, and the result suggest that they were all E. tarda. Six pairs of primers were designed according to six virulence gene published nucleotide sequence, and PCR was developed to detect the distribution of above virulence genes in domestic and oversea isolation strains. There was different in the virulence genes distribution between domestic and oversea isolation strains. We tested the haemolysis of E. tarda, and challenged the mouse and zebrafish using E. tarda CD strain, suggesting that CD strain was virulence.2 Generation of Edwardsiella tarda ghosts by PhiX174 lysis gene EBacterial ghosts may be generated by the controlled expression of the PhiX174 lysis gene E in Gramnegative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The objective of this study was to examine the potential utility of the PhiX174 E gene driven by the PR/cI857 regulatory system for the generation of E. tarda ghosts. The E. tarda ghost (ETG) vaccine was successfully prepared using this technology and tested in safe trials.3 Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infectionThe Edwardsiella tarda ghost (ETG) vaccine was tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease.4 Immune Effect of Edwardsilla tarda ghosts on zebrafishIn this work, we describe a method to assay the phagocytic activity of zebrafish leukocytes induced by E. tarda ghosts (ETG) vaccine. Control groups included zebrafish immunized with formalin-killed E. tarda (FKC) or zebrafish treated with phosphate-buffered saline (PBS), respectively. The results showed that the leukocytes phagocytic activity was significantly higher in ETG group than those in the other two groups. Most important, ETG-immunized fish were significantly protected against E. tarda challenge (83.3% survival) compared to 70 and 6.7% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG vaccine could confer protection against infection in a zebrafish model of disease.5 Edwardsiella tarda Ghost Production by Expression of Lysis Gene E and Staphylococcal Nuclease AA dual vector expressing the ghost-inducing PhiX174 lysis E gene and the bacterial DNA degrading staphylococcal nuclease A (SNA) gene was constructed to solve the problem of remnant antibiotic resistance genes and genomic DNA with intact pathogenic islands in the final product of E. tarda ghosts (ETG). The expression of staphylococcal nuclease A in E. tarda resulted in intracellular accumulation of the protein and degradation of the host DNA into fragments. The dual expression system for the nuclease are presented and were combined with the protein E-mediated lysis system. Under optimized conditions for the coexpression of gene E and the staphylococcal nuclease, the concentration of viable cells fell below the lower limit of detection, whereas the rates of ghost formation were not affected. The 26 amino acid N-terminal sequence of SNA fused with theλphage Cro gene, showed successful degradation of bacterial nucleic acids. BG were generated via coexpression of the SNA gene and lysis gene E under the control of eachλPR promoter. The ghost bacteria generation system we describe is advantageous as it allows the use of a single plasmid, improves safety and vaccine purity by limiting residual genetic content from the ghost bacteria.更多还原