本文關(guān)鍵詞： FMDV 結(jié)構(gòu)蛋白VP1 B細胞表位 T細胞表位 合成肽疫苗　出處：《中國農(nóng)業(yè)科學院》2012年碩士論文　論文類型：學位論文

【摘要】：近年來，國內(nèi)外口蹄疫廣為流行，作為口蹄疫防控主要手段的滅活疫苗由于存在各種弊端，使得口蹄疫新型疫苗的研制成為預(yù)防、控制口蹄疫的必由之路。FMDV呈現(xiàn)七個血清型并在型內(nèi)抗原差異也相對較大。像FMDV這樣高度易變的病毒，其抗原在存在或缺乏免疫選擇壓力下都會發(fā)生變異，預(yù)期其抗原性差異與蛋白氨基酸序列不同相關(guān)。FMDV表達的蛋白包括4種結(jié)構(gòu)蛋白VP1、VP2、VP3和VP4和8種非結(jié)構(gòu)蛋白Lab或Lb，2A，2B，2C，3A，3B，3C和3D。不同血清型的FMDV組成相應(yīng)結(jié)構(gòu)蛋白的氨基酸數(shù)不同，結(jié)構(gòu)蛋白VP1差異最大，其次為VP3和VP2。VP1上氨基酸數(shù)目的不同主要表現(xiàn)在VP1G－H環(huán)內(nèi)RGD序列前后氨基酸的插入和缺失。隨著大量的試驗研究證明VP1G－H環(huán)功能的多樣性，除了有優(yōu)勢抗原性特征外，，且含有與細胞表面整聯(lián)蛋白結(jié)合的Arg-Gly-Asp(RGD)基序。許多研究表位疫苗的試驗，都以含有此基序的表位作為B抗原表位，誘導動物產(chǎn)生中和抗體，且側(cè)翼序列對免疫效果具一定的影響。因此本試驗選擇AsiaⅠ型FMDVVP1129～169序列作為B抗原表位。FMD的免疫機制主要是T細胞依賴性體液免疫應(yīng)答機制，并有研究表明序列中含有T細胞 表位的FMD合成肽疫苗能提高其免疫應(yīng)答效果。FMDV非結(jié)構(gòu)蛋白3D346～370是針對宿主豬的一個不受MHC限制的T細胞表位。合成肽設(shè)計中也多用到通用T細胞表位來改善免疫應(yīng)答效果。如人工合成的PADRE就是應(yīng)用較廣的一個通用T細胞表位。本試驗同時選擇了以上兩個Th表位來優(yōu)化FMDV合成肽疫苗。本研究共設(shè)計了3個合成肽組合，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))。制成的合成肽疫苗與滅活和PBS陰性對照苗分組免疫豚鼠，通過間接ELISA檢測血清IgG、IgG1、IgG2a抗體水平，病毒微量細胞中和試驗檢測中和抗體水平；外周血T淋巴細胞CD4+CD8+亞群流式檢測，豚鼠脾淋巴細胞增殖、淋巴細胞因子測定和小鼠脾淋巴細胞因子ELIspot來評估合成肽疫苗的細胞免疫水平。疫苗免疫豚鼠42d后攻毒保護試驗，結(jié)果表明合成肽P1疫苗可100%保護動物免受感染，合成肽P2疫苗完全不能保護動物，合成肽P3疫苗僅有50%可保護動物不被感染。以上研究結(jié)果為AsiaⅠ型合成肽疫苗的研制提供了可靠的依據(jù)。
[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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