發(fā)布時間：2018-01-20 04:05

  本文關鍵詞： 轉基因兔球蟲 大型艾美耳球蟲 兔瘟病毒 VP60 免疫應答　出處：《中國農(nóng)業(yè)大學》2017年博士論文　論文類型：學位論文

【摘要】：養(yǎng)兔業(yè)的健康發(fā)展受到兔瘟和兔球蟲病的嚴重威脅。兔瘟組織滅活疫苗潛在的生物安全隱患、仔兔的疫苗免疫持續(xù)期短,以及慢性感染家兔的持續(xù)排毒是兔瘟防控需解決的重要問題。同時,兔球蟲具有良好的免疫原性,一次免疫即可激發(fā)較強的黏膜免疫應答。近年來本課題組一直進行以轉基因艾美耳球蟲為載體的活疫苗研究,以期為生產(chǎn)提供更加安全、便捷的免疫策略。因此,本研究以兔球蟲為載體重組表達兔瘟病毒衣殼蛋白VP60-P2亞域,評估轉基因兔球蟲作為重組疫苗載體的可行性,為兔瘟和兔球蟲病新型疫苗的研發(fā)提供思路。我們首先利用染色體步移的方法獲取了大型艾美耳球蟲proflin基因的調控序列,并通過體外轉染證實了其啟動外源基因在兔球蟲表達的能力。但我們未能使用該調控序列實現(xiàn)兔球蟲的穩(wěn)定轉染,故此我們使用柔嫩艾美耳球蟲保守的組蛋白4(His4)基因作為調控序列進行轉染,成功獲得了一株表達黃色、紅色熒光蛋白的轉基因大型艾美耳球蟲(EmagER)。我們觀察到熒光蛋白在球蟲生活史的各個階段均有表達;生物學特性對比試驗結果表明,EmagER與原始蟲株具有相似的繁殖力和免疫原性。為檢測轉基因球蟲激發(fā)宿主免疫應答的能力,我們針對當前家兔免疫學和疫苗學研究工具比較缺乏的現(xiàn)狀,建立了家兔細胞免疫檢測平臺。我們利用qPCR方法對EmagER接種后家兔免疫應答相關的細胞因子轉錄水平進行了檢測。結果顯示EmaER免疫能夠有效激發(fā)家兔腸道部位(腸系膜淋巴結,mesenteric lymphnodes,MLN)的淋巴細胞產(chǎn)生針對外源蛋白的Th-1型細胞免疫應答。此外,我們還制備了針對家兔CD3、CD4、CD8、IFN-γ、TNF-α等免疫分子的單克隆抗體,通過免疫印跡和流式細胞術驗證,獲得了可用于流式細胞術的CD4和TNF-α單克隆抗體。為家兔免疫學研究提供了有力的工具。進而,我們構建了表達兔瘟病毒衣殼蛋白VP60-P2亞域的轉基因大型艾美耳球蟲(EmagE-VP60)。我們發(fā)現(xiàn),在蟲體的裂殖生殖時期,VP60-P2在細胞膜表面和胞漿內同時表達,能夠被宿主免疫系統(tǒng)識別。EmagE-VP60免疫家兔后,針對VP60-P2蛋白的抗體水平和外周血單個核細胞特異性CD8+TNF-α+細胞比例較野生.蟲株免疫組稍有上升但無顯著差異;腸道派爾氏結(payer's patches)、MLN淋巴細胞經(jīng)VP60-P2刺激后,Th-1型細胞因子相對轉錄水平較野生蟲株免疫組顯著上升。這些結果表明,EmagE-VP60表達的VP60-P2能夠在腸道部位激發(fā)宿主產(chǎn)生針對外源蛋白的特異性免疫應答。綜上所述,本研究首次建立了兔艾美耳球蟲的轉染平臺,構建了表達兔瘟病毒抗原的轉基因大型艾美耳球蟲。轉基因球蟲免疫后能夠激發(fā)宿主在腸道部位產(chǎn)生針對外源蛋白的特異性免疫應答,為今后的轉基因兔球蟲活載體疫苗的免疫機制研究和應用研發(fā)提供了良好的基礎。
[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- 緯, TNF- 偽 and other immunomolecules were identified by immunoblotting and flow cytometry. Monoclonal antibodies to CD4 and TNF- 偽 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- 偽 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.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S858.291

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