【作者】 张智慧；

【导师】 张元兴； 吴海珍；

【作者基本信息】 华东理工大学 ， 生物化工， 2013， 博士

【摘要】 由鳗弧菌等病原菌所导致的弧菌病是我国沿海一种常见的水产业传染病,本实验室前期通过基因无标记缺失技术,构建了鳗弧菌减毒活疫苗MVAV6203。在临床应用中,该减毒活疫苗能很好地保护经济鱼种免受鳗弧菌的感染,但其免疫保护的机理还不清楚。而斑马鱼是一种目前广泛应用于研究领域的模式生物,本课题以斑马鱼为动物模型,研究了鳗弧菌MVAV6203的免疫保护机理。首先完成了鳗弧菌减毒活疫苗MVAV6203注射免疫斑马鱼的免疫保护分析,疫苗免疫(剂量为105CFU/尾,5μl/尾)一个月之后的相对免疫保护力达到90%以上,与在牙鲆等经济鱼种上的结果类似,说明斑马鱼经疫苗免疫后有类似的保护作用,可作为疫苗免疫保护机理研究的动物模型。在免疫鱼体血清中特异性抗体的分析中发现,在疫苗注射接种后抗体会持续表达,至免疫后第14天时达到峰值,说明鱼体注射接种该疫苗能激发有效的体液免疫反应。另外,经过免疫的斑马鱼对哈维氏弧菌及创伤弧菌有一定的交叉保护作用,相对保护力都在40%左右,但对副溶血弧菌则没有保护作用。其次,进行了鳗弧菌减毒活疫苗的浸泡免疫效果和应答机理分析。浸泡免疫是鱼用减毒活疫苗常用的接种方式,鳗弧菌减毒活疫苗以1×108CFU/ml浸泡接种斑马鱼,免疫1个月后的攻毒试验发现,免疫组斑马鱼的累积死亡率低于10%,其相对免疫保护力为90%以上,说明该减毒活疫苗能很好地保护斑马鱼免受鳗弧菌的感染。通过对免疫后一个月内斑马鱼血清中鳗弧菌特异性抗体水平的观察发现,在免疫后的4周左右抗体水平明显上调,说明疫苗的免疫保护作用与抗体的产生密切相关。在免疫之后14天内,斑马鱼脾脏和肝脏中包括IL-1β、IL-8、MHCⅠ和MHCⅡ等在内的多种重要免疫相关基因的转录水平有不同程度的上调,说明该减毒活疫苗能有效激发斑马鱼体内的免疫反应,产生免疫记忆并使受免疫的鱼体具有良好的抗病性。进一步对鳗弧菌减毒活疫苗浸泡接种鱼体的进入途径和亲鱼接种免疫后胚胎及幼鱼内母源免疫成份进行了初步研究。以鳗弧菌toxR基因为特异性标记分子,通过PCR方法对浸泡免疫后斑马鱼的鳗弧菌减毒活疫苗存在情况进行了研究。在浸泡免疫后的0.5h内,发现在鳃、后肠和皮肤中均有鳗弧菌的存在,而免疫后0.5h至3h,仅皮肤中仍能检测出鳗弧菌的存在,而研究报道鱼体的皮肤是鳗弧菌感染定殖的主要部位。这一结果提示鳗弧菌减毒活疫苗进入鱼体的方式和激活免疫反应的途径与野生株相似。免疫和未免疫斑马鱼在攻毒后的免疫应答差异分析,是研究疫苗保护机理的重要方法之一。免疫和未免疫斑马鱼在人工攻毒(鳗弧菌强毒株浸泡攻毒,105CFU/ml,20min)后,72h内的抗体产生情况分析发现,免疫斑马鱼在攻毒后的抗体水平变化不明显,这说明相较于特异性抗体数量的变化,接种疫苗对抗体亲和力等性质影响更大。未免疫斑马鱼的抗体水平则在攻毒后的第72h开始上升,说明未免疫的斑马鱼需要一定时间激发特异性抗体。免疫相关基因的转录水平变化分析发现,未免疫斑马鱼中的促炎症类因子如IL-1β等表达明显上调,而免疫斑马鱼的促炎症类因子表达量没有明显增加,特异性免疫应答相关基因MHCⅠ和MHCⅡ等的表达上调。经过免疫的斑马鱼在受到病原感染后能较快地激发适应性免疫系统,同时产生特异性抗体以保护机体,在清除病原的同时能抑制因病原破坏机体组织而产生的炎症发生,而未免疫斑马鱼在清除病原时会产生明显的炎症反应,导致组织损伤甚至鱼体死亡。同时,进行了鳗弧菌减毒活疫苗加强免疫的保护力分析。加强免疫是延长疫苗保护时间,强化疫苗保护力的常用方法。在免疫后8周内的免疫保护效力分析中发现,未进行加免的免疫组相对免疫保护力33%,而单次加免或两次加免的免疫鱼的相对免疫保护力均较高,超过80%,说明加强免疫能延长疫苗对鱼体的保护作用。免疫斑马鱼血清中的特异性抗体水平分析发现,未加免组的斑马鱼抗体水平在疫苗免疫后的6和8周抗体水平明显高于加免组鱼体,即抗体水平与保护力之间未呈对应关系,说明疫苗加免所激发的有效保护与特异性IgM量的高低无直接关联。母源免疫是鱼类免疫的一个重要方面。鳗弧菌MVAV6203注射免疫斑马鱼雌性亲鱼交配后所产的胚胎胞质液中,鳗弧菌特异性IgM的水平相比对照没有明显上升,且胚胎胞质液对鳗弧菌的特异性杀菌活力没有明显变化,但对大肠杆菌的杀菌活性有所上升。SDS-PAGE电泳检测胚胎胞质总蛋白的结果显示免疫雌鱼所产生的胚胎与对照也没有明显变化,Western blotting实验表明免疫组与对照组的胚胎胞质中溶菌酶表达量也无明显差别。转录水平的实验则表明,IL-8与溶菌酶的基因转录启动较早,而在发育早期IgM的mRNA则主要依赖于母源免疫。免疫组和对照组间的比较则显示,接种疫苗与否对上述免疫相关因子的表达影响不大。用鳗弧菌浸泡攻毒免疫组和对照组所产幼鱼后发现,来自免疫组的幼鱼体内鳗弧菌消除速率较快,说明亲鱼免疫后所产生的幼鱼对鳗弧菌有较强的清除能力。对攻毒后幼鱼相关免疫基因的转录水平考察发现,免疫组的先天免疫基因转录水平较对照组的低。鳗弧菌MVAV6203免疫对斑马负母源免疫与发育早期幼鱼的免疫有一定的影响。综上所述,首次利用模式生物斑马鱼为实验动物,系统地研究了鳗弧菌减毒活疫苗的保护机理,对疫苗与鱼体免疫系统之间应答分析有助于鱼用疫苗的进一步开发和应用。更多还原

【Abstract】 Vibrio anguillarum is a main aetiological agent of vibriosis, a common disease along coast of China. A live attenuated vaccine V. anguillarum strain MVAV6203was established in our previous work. This strain was proven to be protective of fish out of vibriosis in some farms. But the mechanism of the immuneprotection is still unclear. In this study, the zebrafish, a powerful model animal in science, was utilized to reveal the mechanism.Firstly, the zebrafish were injected with V. anguillarum MVAV6203(1×105CFU/fish). After4weeks, the zebrafish were challenged with wild-type V. anguillarum MVM425. The vaccinated groups significantly survived from the infection of wild-type V. anguillarum MVM425with about90%RPS, which was similar to the results on industrial fish such as flounder. It is indicated that zebrafish could be used as a model animal to investigate the mechanism of protection. Moreover, the serum specific antibody level was up-regulated following14days post vaccination, which suggested the responses of humoral immunity was triggered. In addition, the vaccination led to cross-protection to Vibrio harveyi and Vibrio vulnificus with about40%RPS, while no protection to Vibrio parahaemolyticus.Secondly, zebrafish were protected against the wild pathogenic strain with remarkable RPS of90%when bath-vaccinated with the live attenuated vaccine (1×108CFU/ml). The specific antibody response of vaccinated zebrafish against V. anguillarum was found to gradually increase during28days post-vaccination. Moreover, the expressions of several immune-related genes including IL-1β、IL-8、MHC and MHC Ⅱ were enhanced in the spleens and livers of zebrafish, which was a symbol of the immune-response from vaccination to emerge the specific protection. In addition, the entrance of the live attenuated V. anguillarum was detected with PCR using toxR gene as a molecule marker after bath-vaccination for the entrance is critical to the administration of the live attenuated vaccine. As shown in the results, the toxR gene was detected in the gills and intestines samples on0.5h post bath-vaccination, which stated the gill and intestine could be the tissues for entrance of V. anguillarum MVAV6203. The gene was also found in the skin samples on0.5-3h post vaccination, which was consistent to the reported founding that skin is vital for the wild-type V. anguillarum to infect fish. It was indicated the intaking with water or the colonization of shin could be the route for the live attenuated V. anguillarum to induce immune responses. Furthermore, the immune responses including specific antibody and immune-related genes expression were investigated to clarify the mechanism responsible for protection in bath-vaccinated and non-vaccinated zebrafish after challenge. As the results, specific antibody response of fish was stronger in non-vaccinated zebrafish than that in vaccinated group in3days post infection. On the aspect of gene transcription, genes encoding pro-inflammatory cytokine and chemokine were much more up-regulated in non-vaccinated group than in vaccinated group after infection. On the contrast, the expression levels of adaptive immune-related genes were enhanced in vaccinated group after challenge. These results suggested that zebrafish vaccinated with the live attenuated vaccine was triggered the protection to avoid hurt by repressing the inflammation and strengthen the adaptive immunity at the early stage of infection.Meanwhile, the effects of booster vaccination with live attenuated V. anguillarum were investigated using bath-vaccination in a zebrafish model. Zebrafish that received booster doses at2weeks or both2and4weeks after primary vaccination were better protected compared to those in the non-booster group in the8weeks post vaccination. In addition, the booster did not induce a stronger specific antibody response. No correlation between a weak specific antibody response and strong protection was observed, indicating the complicated role of IgM in fish immunity.The maternal transfer is also an important aspect of fish immunity. The level of specific IgM against V. anguillarum did not rise in the embryo cytosol at14days post vaccination from the female broodstock injection-vaccinated with V. anguillarum MVAV6203. Meanwhile the bacteriolytic activity to V. anguillarum of the embryo cytosol did not change, but was up-regulated of that to E. coli. With Western blotting, the bands of lysozyme were detected with anti-zebrafish lysozyme monoclonal antibody in both embryo cytosol samples from vaccinated and non-vaccinated broodstock, but there was no difference between the two groups. The transcriptions of IL-8and lysozyme were initiated early in the embryo development. To the contrast, the mRNA of IgM was relied on maternal transfer. The vaccination on broodstock did not impact on the mRNA level of immune-related genes in the embryo. The larvaes at6days post fertilization from vaccinated and non-vaccinated zebrafish broodstock were bath-challenged with V. anguillarum. The V. anguillarum in larvaes from vaccinated group was eliminated more quickly than in non-vaccinated group, which suggests stronger clearness in larvaes from vaccinated broodstock. Meanwhile, the transcriptional levels of genes of innate immune were lower in larvaes from vaccinated zebrafish than those from non-vaccinated zebrafish after challenge. The results on the maternal transfer and the immunity of larvaes indicated that injection-vaccination with V. anguillarum MVAV6203has impact on the zebrafish immunity maternal transfer at14days post vaccination. In conclusion, the mechanism of immuneprotection of live attenuated V. anguillarum was comprehensively investigated using the zebrafish as a model animal. The results will facilitate the further study on the relationship between vaccine and fish immunity.更多还原