本文選題：PEDV　切入點(diǎn)：S1蛋白　出處：《浙江理工大學(xué)》2016年碩士論文

【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是全球流行的極具威脅性的豬病之一,由具有高致死率的豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)引起。該病在世界各地的爆發(fā)給全球養(yǎng)豬業(yè)造成了重大損失,但迄今為止尚無有效安全的疫苗可用。通過新手段研制PEDV疫苗十分必要。S蛋白是PEDV的主要結(jié)構(gòu)抗原蛋白,由S1區(qū)和S2區(qū)組成,其中S1含有3個(gè)中和表位區(qū)域,常作為PEDV基因工程疫苗研究中最重要的一個(gè)靶蛋白。本研究擬構(gòu)建S1基因的系列重組桿狀病毒:rv Ac-S1、rv Ac-SP-S1、rv Ac-SP-S1-TMD、rv Ac-SP-S1-e GFP、rv Ac-SP-S1-e GFP-TMD,以實(shí)現(xiàn)在昆蟲細(xì)胞中高效表達(dá)S1或者在重組桿狀病毒表面展示S1。并將上述S1蛋白及表面展示S1的重組病毒分別作為蛋白亞單位疫苗和病毒活載體疫苗使用,初步探索兩種疫苗在BALB/c小鼠模型上的免疫效果。直接熒光、間接免疫熒光、免疫印跡、免疫電鏡對S1的表達(dá)定位進(jìn)行分析。結(jié)果表明:rv Ac-SP-S1-e GFP和rv Ac-SP-S1-e GFP-TMD感染Sf9細(xì)胞后,S1在Sf9細(xì)胞中高效表達(dá),且S1胞內(nèi)表達(dá)量前者大于后者;S1定位于感染細(xì)胞的細(xì)胞膜附近及rv Ac-SP-S1-e GFP-TMD表面。制備S1重組蛋白免疫原及表面展示S1的重組病毒顆粒性免疫原皮下免疫小鼠。間接ELISA、微量細(xì)胞培養(yǎng)中和試驗(yàn)、脾淋巴細(xì)胞增殖試驗(yàn)對免疫原刺激小鼠產(chǎn)生的體液免疫及細(xì)胞免疫應(yīng)答效果進(jìn)行檢測。結(jié)果表明:展示S1的重組桿狀病毒免疫血清中存在特異性抗體,抗體效價(jià)達(dá)1:5000,并具有一定中和PEDV能力,中和效價(jià)達(dá)1:26。在PEDV刺激原作用下,展示S1重組桿狀病毒免疫鼠的脾淋巴細(xì)胞與對照組相比增殖顯著(P0.05)。而S1重組蛋白免疫原小鼠后未取得理想效果。上述可知,表面展示S1的rv Ac-SP-S1-e GFP-TMD重組桿狀病毒直接免疫小鼠可誘導(dǎo)產(chǎn)生體液及細(xì)胞免疫反應(yīng)。本研究首次將PEDV CV777全長S1蛋白表達(dá)于昆蟲細(xì)胞及展示于桿狀病毒表面以用于PEDV疫苗研發(fā),并取得一定初步成果,為PEDV新型疫苗的研發(fā)奠定了基礎(chǔ)。
[Abstract]:Porcine epidemic diarrhea (PED) is one of the most threatening swine diseases in the world, caused by porcine epidemic diarrhea virus (PEDVV). However, there is no effective and safe vaccine available so far. It is necessary to develop PEDV vaccine by new means. S protein is the main structural antigen protein of PEDV, which consists of S1 region and S2 region, among which S1 contains three neutralization epitopes. This study is intended to construct a series of recombinant baculovirus of S1 gene: rv Ac-SP-S1rv rv Ac-SP-S1-TMDrv Ac-SP-S1-e GFPrv Ac-SP-S1-e GFP-TMD.The aim of this study is to achieve high expression of S1 in insect cells or recombinant baculovirus in insect cells. Virus surface display S1. And the above S1 protein and surface display S1 recombinant virus as protein subunit vaccine and live vector vaccine, To explore the immunological effects of the two vaccines on BALB/c mice model. Direct fluorescence, indirect immunofluorescence, immunoblot, The expression localization of S1 was analyzed by immuno-electron microscope. The results showed that the expression of S1 was highly expressed in Sf9 cells after the infection of Sf9 cells with Sf9 GFP and rv Ac-SP-S1-e GFP-TMD. The intracellular expression of S1 was higher than that of the latter. S1 was located near the cell membrane of infected cells and on the surface of rv Ac-SP-S1-e GFP-TMD. The immunogen of S1 recombinant protein and the recombinant viral granulocytic immunogen with S1 on the surface were prepared and subcutaneously immunized mice with S1. Elsa, microcell culture neutralization test, Spleen lymphocyte proliferation test was used to detect the humoral immunity and cellular immune response induced by immunogen in mice. The results showed that there were specific antibodies in S1 recombinant baculovirus immune serum. The titer of the antibody was 1: 5000and had the ability to neutralize PEDV, and the neutralization titer was 1: 26.The antibody was stimulated by PEDV. The spleen lymphocytes of mice immunized with S1 recombinant baculovirus were significantly increased compared with those of the control group (P 0.05). However, the mice immunized with S1 recombinant protein did not achieve satisfactory results. Rv Ac-SP-S1-e GFP-TMD directly immunized mice with S1 on the surface can induce humoral and cellular immune responses. In this study, the full-length S1 protein of PEDV CV777 was first expressed in insect cells and exhibited on the surface of baculovirus for the development of PEDV vaccine. Some preliminary results were obtained, which laid a foundation for the research and development of new PEDV vaccine.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S852.65

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