基于S1蛋白核心中和表位区的猪流行性腹泻亚单位疫苗研究

发布时间：2018-05-31 10:46

本文选题：PED + 毕赤酵母　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：猪流行性腹泻(PED)是由猪流行性腹泻病毒(PEDV)引起的一种猪的急性、高度接触性肠道传染病,主要特征为腹泻、呕吐、脱水和高死亡率。自2010年以来,以PEDV变异株为主引起的PED在我国各省份广泛流行,对我国养猪业造成了严重的经济损失。疫苗免疫是目前防控PED的最有效途径,目前市场主要预防PED的疫苗为多联灭活、或弱毒疫苗,以上疫苗存在保护率低、安全性低、成本昂贵等缺点,未对PED实现有限地防控和净化,市场亟待开发更加安全、经济、高效的新型疫苗(尤其是亚单位疫苗)用于PED的防制。PEDV S1结构域是介导PED保护性免疫的主要功能区,核心中和表位(COE)是S1的最重要功能区,也是目前用于PED亚单位疫苗研究的最小保护抗原。因此,本研究通过毕赤酵母表达系统对PEDV S1的COE进行表达。同时尝试将COE与人和猪的Ig G1 Fc进行融合来提高COE抗原的免疫效果,取得的主要结果如下:a)利用PCR分别扩增出COE、P1Fc、H1Fc片段,通过重叠PCR技术拼接COE-P1Fc和COE-H1Fc片段,成功构建了酵母表达载体p PICZαA-COE、p PICZαA-COEP1Fc和p PICZαA-COE-H1Fc;b)利用酵母表达系统分泌表达产物经Western blotting鉴定,结果表明成功的表达了r COE、r COE-P1Fc、r COE-H1Fc 3种蛋白。c)免疫小鼠42d后采集各组血清通过间接ELASA和中和试验评价机体产生抗体情况。间接ELASA结果表明3组实验组都产生了较高水平的抗体。血清中和试验结果显示r COE、r COE-P1Fc、r COE-H1Fc实验组中和效价依次为1:160、1:640、1:512,r COE-P1Fc、r COE-H1Fc实验组中和效价显著高于r COE实验组和PBS对照组,结果表明在COE基因基础上融合佐剂P1Fc、H1Fc,明显提高了PED亚单位疫苗效果。本研究首次利用毕赤酵母表达系统表达S1蛋白核心中和表位融合蛋白,对研究PED亚单位疫苗进行首次尝试,且Ig G1 Fc片段具有佐剂功能,能提高机体免疫力,成为防控PED的新型亚单位疫苗。
[Abstract]:Porcine epidemic diarrhea (PED) is an acute, highly contact intestinal infection caused by porcine epidemic diarrhea virus (PEDVV), characterized by diarrhea, vomiting, dehydration and high mortality. Since 2010, PED, mainly caused by PEDV variant strains, has been widely prevalent in various provinces of China, which has caused serious economic losses to the pig industry in China. Vaccine immunization is the most effective way to prevent and control PED at present. At present, the main vaccine to prevent PED is multiplex inactivation or attenuated virus vaccine. These vaccines have the disadvantages of low protection rate, low safety, high cost and so on. In the absence of limited control and purification of PED, it is urgent for the market to develop more safe, economical and efficient new vaccines (especially subunit vaccines) for PED control. PEDV S1 domain is the main functional region that mediates the protective immunity of PED. Core neutralizing epitope (Coe) is the most important functional region of S1 and the least protective antigen for PED subunit vaccine research. Therefore, the COE of PEDV S1 was expressed by Pichia pastoris expression system. At the same time, we try to improve the immune effect of COE antigen by fusion of COE with human and pig IgG1 FC. The main results are as follows: (1) using PCR to amplify COEN P1FcH1 Fc fragment, COE-P1Fc and COE-H1Fc fragments were spliced by overlapping PCR technique. Yeast expression vectors, p PICZ 伪 A-COEG, p PICZ 伪 A-COEP1Fc and p PICZ 伪 A-COE-H1 Fcb, were successfully constructed. The expressed products were identified by Western blotting using yeast expression system. The results showed that the mice were immunized with rCOEE-P1Fcnr COE-H1Fc successfully for 42 days. After immunization, the sera of each group were collected to evaluate the antibody production by indirect ELASA and neutralization test. Indirect ELASA results showed that the three experimental groups produced a higher level of antibodies. The results of serum neutralization test showed that the neutralization titer of the experimental group was 1: 160, 1: 640,1: 512rCOE-P1Fccr COE-H1Fc, and the neutralization titer of the experimental group was significantly higher than that of the experimental group of r COE and the control group of PBS, and the neutralization titer of the experimental group was significantly higher than that of the experimental group of r COE and the control group of PBS. The results showed that the fusion of the adjuvant P1Fc1 Fc1 Fc on the basis of COE gene significantly improved the effect of PED subunit vaccine. In this study, we used Pichia pastoris expression system to express S1 protein core neutralizing epitope fusion protein for the first time, and we tried to study PED subunit vaccine for the first time, and Ig G1 FC fragment has adjuvant function and can improve body immunity. To become a new subunit vaccine for the prevention and control of PED.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.28

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