口蹄疫Asia I型合成肽疫苗的研究

发布时间：2018-01-22 15:58

  本文关键词： FMDV 结构蛋白VP1 B细胞表位 T细胞表位 合成肽疫苗　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】：近年来，国内外口蹄疫广为流行，作为口蹄疫防控主要手段的灭活疫苗由于存在各种弊端，使得口蹄疫新型疫苗的研制成为预防、控制口蹄疫的必由之路。FMDV呈现七个血清型并在型内抗原差异也相对较大。像FMDV这样高度易变的病毒，其抗原在存在或缺乏免疫选择压力下都会发生变异，预期其抗原性差异与蛋白氨基酸序列不同相关。FMDV表达的蛋白包括4种结构蛋白VP1、VP2、VP3和VP4和8种非结构蛋白Lab或Lb，2A，2B，2C，3A，3B，3C和3D。不同血清型的FMDV组成相应结构蛋白的氨基酸数不同，结构蛋白VP1差异最大，其次为VP3和VP2。VP1上氨基酸数目的不同主要表现在VP1G－H环内RGD序列前后氨基酸的插入和缺失。随着大量的试验研究证明VP1G－H环功能的多样性，除了有优势抗原性特征外，，且含有与细胞表面整联蛋白结合的Arg-Gly-Asp(RGD)基序。许多研究表位疫苗的试验，都以含有此基序的表位作为B抗原表位，诱导动物产生中和抗体，且侧翼序列对免疫效果具一定的影响。因此本试验选择AsiaⅠ型FMDVVP1129～169序列作为B抗原表位。FMD的免疫机制主要是T细胞依赖性体液免疫应答机制，并有研究表明序列中含有T细胞 表位的FMD合成肽疫苗能提高其免疫应答效果。FMDV非结构蛋白3D346～370是针对宿主猪的一个不受MHC限制的T细胞表位。合成肽设计中也多用到通用T细胞表位来改善免疫应答效果。如人工合成的PADRE就是应用较广的一个通用T细胞表位。本试验同时选择了以上两个Th表位来优化FMDV合成肽疫苗。本研究共设计了3个合成肽组合，P1：(Ac-VP1_(134~159))4-KKK-3D_(346~370)-KK，P2： Ac-3D_(346~370)-GG-VP1_(129~169)(134(T→C),(RGD→RDD),155(N→D),156(R→C))，P3：Ac-PADRE-GG-VP1_(129~169)(134(T→C),(HGD→RDD),156(R→C))。制成的合成肽疫苗与灭活和PBS阴性对照苗分组免疫豚鼠，通过间接ELISA检测血清IgG、IgG1、IgG2a抗体水平，病毒微量细胞中和试验检测中和抗体水平；外周血T淋巴细胞CD4+CD8+亚群流式检测，豚鼠脾淋巴细胞增殖、淋巴细胞因子测定和小鼠脾淋巴细胞因子ELIspot来评估合成肽疫苗的细胞免疫水平。疫苗免疫豚鼠42d后攻毒保护试验，结果表明合成肽P1疫苗可100%保护动物免受感染，合成肽P2疫苗完全不能保护动物，合成肽P3疫苗仅有50%可保护动物不被感染。以上研究结果为AsiaⅠ型合成肽疫苗的研制提供了可靠的依据。
[Abstract]:In recent years, foot-and-mouth disease is widely prevalent at home and abroad. The inactivated vaccine, which is the main means of prevention and control of foot-and-mouth disease, has a variety of drawbacks, which makes the development of a new vaccine for foot-and-mouth disease prevention. The only way to control foot-and-mouth disease. FMDV presents seven serotypes and there are relatively large differences in intratype antigens. A highly variable virus such as FMDV. The antigenicity of the antigenicity is different from the amino acid sequence of protein. The proteins expressed by FMDV include four structural proteins: VP1and VP2. VP3 and VP4 together with eight nonstructural proteins Lab or Lb2A2A2C3A3A3BH3B3 C and 3D. the amino acids of the corresponding structural proteins were different among different serotypes of FMDV. The difference of structural protein VP1 was greatest. Secondly, the difference in the number of amino acids between VP3 and VP2.VP1 is mainly manifested in the insertion and deletion of amino acids before and after RGD sequence in the VP1G-H ring. The diversity of ring functions. In addition to the characteristics of dominant antigenicity, it also contains the Arg-Gly-Aspen RGD motif which binds to cell surface integrin. Many studies on epitope vaccines have been carried out. The epitopes containing this motif were used as B antigen epitopes to induce the production of neutralizing antibodies in animals. And flanking sequence has a certain effect on the immune response. Therefore, T cell dependence is the main immune mechanism of Asia type 鈪

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