口蹄疫病毒非结构蛋白3D的分段表达及其免疫学功能研究

发布时间：2018-01-10 05:13

本文关键词：口蹄疫病毒非结构蛋白3D的分段表达及其免疫学功能研究　出处：《中国农业科学院》2012年硕士论文　论文类型：学位论文

【摘要】：口蹄疫（Foot-and-mouth disease，FMD）是由口蹄疫病毒（Foot-and-mouth disease virus，FMDV）引起的偶蹄动物的一种急性、热性、高度接触性传染病。一旦发生疫情损失重大，影响恶劣。应用疫苗进行免疫接种是预防、控制乃至消灭FMD的有效手段之一。由于目前使用的灭活疫苗存在诸多缺点，发达国家曾一度停止了该疫苗的使用。近年来出现了多种新型高效FMD分子疫苗，但低免疫原性仍然是制约FMD分子疫苗发展的技术瓶颈。本研究以本实验室前期的研究结果为基础，进一步研究FMDV非结构蛋白3D的免疫增强功能，为研制高效、安全的FMD新型分子疫苗提供理论依据。首先采用生物信息学软件对Asia1型FMDV非结构蛋白3D的T细胞表位进行了预测，根据T细胞表位富集度，，将3D基因分成三段，并设计特异性引物进行PCR扩增，并实现重组蛋白3D1，3D2和3D3在大肠杆菌细胞BL21(DE3)进行表达及纯化。重组蛋白3D1，3D2和3D3定量后分别与等体积的佐剂混合乳化；或分别与表达的Asia1型FMDV多表位重组按照1：2(w/w)混合，并与等体积佐剂混合乳化制备成疫苗。用间接ELISA，流式细胞术、淋巴细胞增殖试验以及ELISPOT等对免疫小鼠血清抗体、脾淋巴细胞增殖能力以及关键细胞因子表达水平进行检测。Western blotting结果显示3D1重组蛋白不能与FMDV阳性血清发生免疫反应外，其他2种蛋白均能与阳性血清发生免疫反应。但三种蛋白都能与抗His单抗发生免疫反应。免疫小鼠血清抗体随着免疫时间的延长持续升高，且3D2或3D3与Asia1型FMDV表位重组抗原联合免疫优于3D1与Asia1型FMDV表位重组抗原。淋巴细胞增殖试验结果显示，3D1与Asia1型FMDV表位重组抗原优于3D2或3D3与Asia1型FMDV表位重组抗原联合免疫。ELISPOT结果表明，重组蛋白3D1，3D2和3D3分别与表位重组抗原组合，分泌IFN-γ的脾淋巴细胞显著高于Asia1型FMDV多表位重组抗原单独免疫组，但遗憾的是，分泌IL-4细胞因子的淋巴细胞各组差异不显著。为了进一步验证三个蛋白的免疫功能，将重组蛋白3D1，3D2和3D3分别与表达的Asia1型FMDV多表位重组按照1：2(w/w)混合或者单独Asia1型FMDV多表位混合制备的疫苗免疫豚鼠，并于加强免疫后14天用500ID50/0.2ml的Asia1型FMDV攻击实验动物，结果显示，重组蛋白3D1、3D2和3D3分别与Asia1型FMDV多表位重组抗原联合免疫组中均有3/4（75%）完全保护，而Asia1型FMDV多表位重组抗原单独免疫组只有2/4动物（50%）完全保护。为了验证三个蛋白对O型FMDV多表位重组抗原的免疫调节功能，将3D1、3D2和3D3分别与猪O型FMDV广谱多表位重组抗原联合或单独3D蛋白免疫猪。液相阻断ELISA（LPB-ELISA）结果显示，在首免后17天，与O型FMDV广谱多表位重组抗原单独免疫组相比，添加3D1、3D2和3D3的均能产生较高水平的特异性抗体，但加强免疫后所有组别间抗体水平没有明显的差异。综上所述，本研究表明表达的三个蛋白，尤其是3D1和3D3，不仅能够改善FMDV表位重组抗原的免疫原性，而且能诱导细胞免疫反应，是一种很好的疫苗佐剂。
[Abstract]:Foot - and - mouth disease ( FMDV ) is an acute , hot and highly contagious disease caused by Foot - and - mouth disease virus ( FMDV ) . It is one of the most effective ways to prevent , control and eliminate the disease . The recombinant protein 3D1 , 3D2 and 3D3 were combined with the recombinant antigen of FMDV . The results showed that the recombinant proteins 3D1 , 3D2 and 3D3 were combined with the recombinant antigen of FMDV . The results of Western blotting showed that the recombinant proteins 3D1 , 3D2 and 3D3 could not react with the recombinant antigen of FMDV epitope . The results showed that the recombinant proteins 3D1 , 3D2 and 3D3 were significantly higher than those of Asia1 type FMDV epitope . In order to further verify the immune function of the three proteins , the recombinant proteins 3D1 , 3D2 and 3D3 were mixed with the expressed Asia1 type FMDV multi - epitope recombination according to 1 : 2 ( w / w ) or single Asia1 type FMDV multiple epitopes . The results showed that the recombinant proteins 3D1 , 3D2 and 3D3 were completely protected by 3 / 4 ( 75 % ) of the Asia1 type FMDV multi - epitope recombinant antigen combined immunization group , and only 2 / 4 animals ( 50 % ) in the Asia1 type FMDV epitope recombinant antigen were completely protected . In order to verify the immunoregulatory function of three proteins against O - type FMDV multi - epitope recombinant antigen , 3D1 , 3D2 and 3D3 were respectively combined with porcine O - type FMDV broad - spectrum multi - epitope recombinant antigen or single 3D protein immunized pigs . In conclusion , the three proteins expressed in this study , especially 3D1 and 3D3 , can not only improve the immunogenicity of FMDV epitope recombinant antigen , but also induce cellular immune response , which is a good vaccine adjuvant .

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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