本文關(guān)鍵詞： 轉(zhuǎn)基因兔球蟲 大型艾美耳球蟲 兔瘟病毒 VP60 免疫應(yīng)答　出處：《中國農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)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(yán)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(shí),兔球蟲具有良好的免疫原性,一次免疫即可激發(fā)較強(qiáng)的黏膜免疫應(yīng)答。近年來本課題組一直進(jìn)行以轉(zhuǎn)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(dá)兔瘟病毒衣殼蛋白VP60-P2亞域,評(píng)估轉(zhuǎn)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(diào)控序列,并通過體外轉(zhuǎn)染證實(shí)了其啟動(dòng)外源基因在兔球蟲表達(dá)的能力。但我們未能使用該調(diào)控序列實(shí)現(xiàn)兔球蟲的穩(wěn)定轉(zhuǎn)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(diào)控序列進(jìn)行轉(zhuǎn)染,成功獲得了一株表達(dá)黃色、紅色熒光蛋白的轉(zhuǎn)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(gè)階段均有表達(dá);生物學(xué)特性對(duì)比試驗(yàn)結(jié)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(cè)轉(zhuǎn)基因球蟲激發(fā)宿主免疫應(yīng)答的能力,我們針對(duì)當(dāng)前家兔免疫學(xué)和疫苗學(xué)研究工具比較缺乏的現(xiàn)狀,建立了家兔細(xì)胞免疫檢測(cè)平臺(tái)。我們利用qPCR方法對(duì)EmagER接種后家兔免疫應(yīng)答相關(guān)的細(xì)胞因子轉(zhuǎn)錄水平進(jìn)行了檢測(cè)。結(jié)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(jié),mesenteric lymphnodes,MLN)的淋巴細(xì)胞產(chǎn)生針對(duì)外源蛋白的Th-1型細(xì)胞免疫應(yīng)答。此外,我們還制備了針對(duì)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(xì)胞術(shù)驗(yàn)證,獲得了可用于流式細(xì)胞術(shù)的CD4和TNF-α單克隆抗體。為家兔免疫學(xué)研究提供了有力的工具。進(jìn)而,我們構(gòu)建了表達(dá)兔瘟病毒衣殼蛋白VP60-P2亞域的轉(zhuǎn)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(shí)期,VP60-P2在細(xì)胞膜表面和胞漿內(nèi)同時(shí)表達(dá),能夠被宿主免疫系統(tǒng)識(shí)別。EmagE-VP60免疫家兔后,針對(duì)VP60-P2蛋白的抗體水平和外周血單個(gè)核細(xì)胞特異性CD8+TNF-α+細(xì)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(jié)(payer's patches)、MLN淋巴細(xì)胞經(jīng)VP60-P2刺激后,Th-1型細(xì)胞因子相對(duì)轉(zhuǎn)錄水平較野生蟲株免疫組顯著上升。這些結(jié)果表明,EmagE-VP60表達(dá)的VP60-P2能夠在腸道部位激發(fā)宿主產(chǎn)生針對(duì)外源蛋白的特異性免疫應(yīng)答。綜上所述,本研究首次建立了兔艾美耳球蟲的轉(zhuǎn)染平臺(tái),構(gòu)建了表達(dá)兔瘟病毒抗原的轉(zhuǎn)基因大型艾美耳球蟲。轉(zhuǎn)基因球蟲免疫后能夠激發(fā)宿主在腸道部位產(chǎn)生針對(duì)外源蛋白的特異性免疫應(yīng)答,為今后的轉(zhuǎn)基因兔球蟲活載體疫苗的免疫機(jī)制研究和應(yīng)用研發(fā)提供了良好的基礎(chǔ)。
[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(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.291

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王荷貴;;淺談當(dāng)前兔瘟流行特點(diǎn)與預(yù)防措施[J];中國畜牧獸醫(yī)文摘;2016年05期

2 毛曉琴;王曉軍;毛世香;;兔瘟病的臨床癥狀及防治[J];畜禽業(yè);2016年03期

3 陶鴿如;劉賢勇;索勛;;常用抗兔球蟲藥物的應(yīng)用及問題分析[J];寄生蟲與醫(yī)學(xué)昆蟲學(xué)報(bào);2014年04期

4 王天有;豐蘭竹;;仔豬磺胺類藥物中毒的診斷[J];養(yǎng)豬;2014年03期

5 謝永平;何玉珍;周沛;馮世文;唐林生;魏曉瑞;馬春霞;;兔病毒性出血癥自然感染病例的解剖病理學(xué)觀察[J];中國畜牧獸醫(yī);2013年02期

6 吳強(qiáng);;淺析兔瘟免疫失敗的原因及對(duì)策[J];中國養(yǎng)兔;2012年07期

7 閆文朝;王天奇;索勛;崔平;顧小龍;韓利方;薛幫群;;家兔球蟲病的研究進(jìn)展[J];中國獸醫(yī)科學(xué);2010年11期

8 李峰;楊慧;苗立中;沈志強(qiáng);;兔病毒性出血癥的診治[J];中國動(dòng)物檢疫;2008年11期

9 闞慶華;王啟明;;大型集約化兔場(chǎng)常見、多發(fā)病的防治及存在的問題[J];中國養(yǎng)兔雜志;2008年07期

10 周碧君,劉剛,曾新華,王安寶;兔瘟的診斷及感染臟器含毒量的測(cè)定[J];貴州農(nóng)業(yè)科學(xué);2000年06期

相關(guān)博士學(xué)位論文 前1條

1 秦梅;融合表達(dá)IgY/IgG Fc片段和H9N2禽流感病毒HA1轉(zhuǎn)基因艾美耳球蟲的構(gòu)建及免疫原性研究[D];中國農(nóng)業(yè)大學(xué);2015年

，

本文編號(hào)：1446776