表达兔瘟病毒VP60-P2的转基因兔球虫激发特异性免疫应答的研究

发布时间：2018-01-20 04:05

本文关键词： 转基因兔球虫大型艾美耳球虫兔瘟病毒 VP60 免疫应答　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：养兔业的健康发展受到兔瘟和兔球虫病的严重威胁。兔瘟组织灭活疫苗潜在的生物安全隐患、仔兔的疫苗免疫持续期短,以及慢性感染家兔的持续排毒是兔瘟防控需解决的重要问题。同时,兔球虫具有良好的免疫原性,一次免疫即可激发较强的黏膜免疫应答。近年来本课题组一直进行以转基因艾美耳球虫为载体的活疫苗研究,以期为生产提供更加安全、便捷的免疫策略。因此,本研究以兔球虫为载体重组表达兔瘟病毒衣壳蛋白VP60-P2亚域,评估转基因兔球虫作为重组疫苗载体的可行性,为兔瘟和兔球虫病新型疫苗的研发提供思路。我们首先利用染色体步移的方法获取了大型艾美耳球虫proflin基因的调控序列,并通过体外转染证实了其启动外源基因在兔球虫表达的能力。但我们未能使用该调控序列实现兔球虫的稳定转染,故此我们使用柔嫩艾美耳球虫保守的组蛋白4(His4)基因作为调控序列进行转染,成功获得了一株表达黄色、红色荧光蛋白的转基因大型艾美耳球虫(EmagER)。我们观察到荧光蛋白在球虫生活史的各个阶段均有表达;生物学特性对比试验结果表明,EmagER与原始虫株具有相似的繁殖力和免疫原性。为检测转基因球虫激发宿主免疫应答的能力,我们针对当前家兔免疫学和疫苗学研究工具比较缺乏的现状,建立了家兔细胞免疫检测平台。我们利用qPCR方法对EmagER接种后家兔免疫应答相关的细胞因子转录水平进行了检测。结果显示EmaER免疫能够有效激发家兔肠道部位(肠系膜淋巴结,mesenteric lymphnodes,MLN)的淋巴细胞产生针对外源蛋白的Th-1型细胞免疫应答。此外,我们还制备了针对家兔CD3、CD4、CD8、IFN-γ、TNF-α等免疫分子的单克隆抗体,通过免疫印迹和流式细胞术验证,获得了可用于流式细胞术的CD4和TNF-α单克隆抗体。为家兔免疫学研究提供了有力的工具。进而,我们构建了表达兔瘟病毒衣壳蛋白VP60-P2亚域的转基因大型艾美耳球虫(EmagE-VP60)。我们发现,在虫体的裂殖生殖时期,VP60-P2在细胞膜表面和胞浆内同时表达,能够被宿主免疫系统识别。EmagE-VP60免疫家兔后,针对VP60-P2蛋白的抗体水平和外周血单个核细胞特异性CD8+TNF-α+细胞比例较野生.虫株免疫组稍有上升但无显著差异;肠道派尔氏结(payer's patches)、MLN淋巴细胞经VP60-P2刺激后,Th-1型细胞因子相对转录水平较野生虫株免疫组显著上升。这些结果表明,EmagE-VP60表达的VP60-P2能够在肠道部位激发宿主产生针对外源蛋白的特异性免疫应答。综上所述,本研究首次建立了兔艾美耳球虫的转染平台,构建了表达兔瘟病毒抗原的转基因大型艾美耳球虫。转基因球虫免疫后能够激发宿主在肠道部位产生针对外源蛋白的特异性免疫应答,为今后的转基因兔球虫活载体疫苗的免疫机制研究和应用研发提供了良好的基础。
[Abstract]:The healthy development of rabbit industry is seriously threatened by rabbit plague and rabbit coccidiosis. And the continuous detoxification of chronic infection rabbits is an important problem to be solved in the prevention and control of rabbit plague. At the same time, the rabbit coccidia has good immunogenicity. In recent years, our team has been carrying out live vaccine research with transgenic Eimeria japonica as vector, in order to provide more safety for production. Therefore, in order to evaluate the feasibility of transgenic rabbit coccidia as recombinant vaccine vector, the recombinant expression of capsid protein VP60-P2 subdomain of rabbit plague virus was studied. To provide ideas for the development of new vaccines against rabbit plague and rabbit coccidiosis, we first obtained the regulatory sequence of proflin gene of Eimeria macrophylla by the method of chromosome step. The ability to initiate the expression of exogenous genes in rabbit coccidiosis was confirmed by transfection in vitro, but we could not use this regulatory sequence to achieve stable transfection of coccidiosis. Therefore, we used the conserved histone 4 His4) gene of Eimeria tenella as the regulatory sequence for transfection, and successfully obtained a yellow expression strain. We observed that the fluorescent protein was expressed at all stages of the life cycle of Emagerus. The comparison of biological characteristics showed that EmagER had similar fecundity and immunogenicity with the original strain, and was used to detect the ability of transgenic coccidia to stimulate host immune response. We aim at the lack of immunology and vaccine research tools in rabbits. A rabbit cellular immunoassay platform was established. We used qPCR method to detect the cytokine transcription level related to immune response after EmagER inoculation in rabbits. The results showed that EmaER immunization could be used to detect cytokine transcription in rabbits. Effective stimulation of intestinal tract in rabbits (. Mesenteric lymph nodes. Mesenteric lymphocytes produced Th-1 type cellular immune response to foreign proteins. In addition, we also prepared rabbit CD3. Monoclonal antibodies against CD4, CD8, IFN- 纬, TNF- 伪 and other immunomolecules were identified by immunoblotting and flow cytometry. Monoclonal antibodies to CD4 and TNF- 伪 were obtained for flow cytometry, which provided a powerful tool for immunological study of rabbits. A transgenic EmagE-VP60 was constructed to express the VP60-P2 subdomain of rabbit plague virus capsid protein. VP60-P2 was expressed simultaneously on the surface of the cell membrane and in the cytoplasm, and could be recognized by the host immune system. EmagE-VP60 immunized rabbits. The antibody level of VP60-P2 protein and the proportion of specific CD8 TNF- 伪 cells in peripheral blood mononuclear cells were higher than those in wild ones. VP60-P2 was used to stimulate the MLN lymphocytes in Paier's node of intestinal tract. The relative transcription level of Th-1 cytokines was significantly higher than that of wild insect strains. The VP60-P2 expressed by EmagE-VP60 can stimulate the host to produce a specific immune response to exogenous proteins in the intestinal tract. In this study, the transfection platform of Eimeria rabbits was established for the first time. Transgenic Escherichia coli was constructed to express rabbit plague virus antigen. The transgenic coccidia could stimulate the host to produce a specific immune response to foreign proteins in the intestinal tract after immunization. It provides a good basis for the study of immune mechanism and application research and development of transgenic rabbit coccidia live vector vaccine in the future.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S858.291

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