杆状病毒表达系统表达猪流行性腹泻病毒S1蛋白及其免疫原性的研究

发布时间：2018-03-29 23:25

本文选题：PEDV　切入点：S1蛋白　出处：《浙江理工大学》2016年硕士论文

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是全球流行的极具威胁性的猪病之一,由具有高致死率的猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起。该病在世界各地的爆发给全球养猪业造成了重大损失,但迄今为止尚无有效安全的疫苗可用。通过新手段研制PEDV疫苗十分必要。S蛋白是PEDV的主要结构抗原蛋白,由S1区和S2区组成,其中S1含有3个中和表位区域,常作为PEDV基因工程疫苗研究中最重要的一个靶蛋白。本研究拟构建S1基因的系列重组杆状病毒: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,以实现在昆虫细胞中高效表达S1或者在重组杆状病毒表面展示S1。并将上述S1蛋白及表面展示S1的重组病毒分别作为蛋白亚单位疫苗和病毒活载体疫苗使用,初步探索两种疫苗在BALB/c小鼠模型上的免疫效果。直接荧光、间接免疫荧光、免疫印迹、免疫电镜对S1的表达定位进行分析。结果表明:rv Ac-SP-S1-e GFP和rv Ac-SP-S1-e GFP-TMD感染Sf9细胞后,S1在Sf9细胞中高效表达,且S1胞内表达量前者大于后者;S1定位于感染细胞的细胞膜附近及rv Ac-SP-S1-e GFP-TMD表面。制备S1重组蛋白免疫原及表面展示S1的重组病毒颗粒性免疫原皮下免疫小鼠。间接ELISA、微量细胞培养中和试验、脾淋巴细胞增殖试验对免疫原刺激小鼠产生的体液免疫及细胞免疫应答效果进行检测。结果表明:展示S1的重组杆状病毒免疫血清中存在特异性抗体,抗体效价达1:5000,并具有一定中和PEDV能力,中和效价达1:26。在PEDV刺激原作用下,展示S1重组杆状病毒免疫鼠的脾淋巴细胞与对照组相比增殖显著(P0.05)。而S1重组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重组杆状病毒直接免疫小鼠可诱导产生体液及细胞免疫反应。本研究首次将PEDV CV777全长S1蛋白表达于昆虫细胞及展示于杆状病毒表面以用于PEDV疫苗研发,并取得一定初步成果,为PEDV新型疫苗的研发奠定了基础。
[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.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.65

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