【作者】 刘新生；

【导师】 王永录；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 硕士

【摘要】 表位肽疫苗作为一种新型疫苗,已成为口蹄疫(Foot-and-Mouth Disease,FMD)疫苗研究的热点之一。由于细胞免疫在口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)的免疫应答中发挥着重要作用,而结构蛋白VP1内又包含着FMDV的主要抗原位点。因此,筛选和鉴定VP1蛋白有效的H-2Ld限制性T细胞抗原表位,不仅可为进一步研究针对FMDV自然宿主的T细胞抗原表位提供方法和依据,也可为进一步研制FMDV表位肽疫苗奠定基础。本研究通过扩增并测序,获得了完整的FMDV AF/72株VP1序列。采用密码子优化表达方法,在大肠杆菌中高效表达了该VP1蛋白。经过SDS-PAGE电泳及Western Blotting检测,其分子量与预期大小相符合,并且保持了良好的免疫原性。与此同时,利用相关生物信息学软件对VP1中潜在的H-2Dd、H-2Kd、H-2Ld限制性T细胞表位进行了预测分析,初步筛选并合成了30个10肽片段作为候选表位肽段。将VP1蛋白免疫BALB/c小鼠,制备经VP1蛋白致敏的脾淋巴细胞,与30个候选表位肽段分别进行体外共培养刺激,经淋巴细胞增殖实验和γ干扰素ELISPOT实验,鉴定出2个H-2d限制性T细胞表位,即H-2Kd限制性T细胞表位pK1(AYHKGPFTRL)和H-2Dd限制性T细胞表位pD7(TGESADPVTT)。本研究筛选并鉴定了FMDV AF/72株结构蛋白VP1中的H-2d限制性T细胞表位,为FMDV T细胞表位的研究提供方法和依据,为进一步研制FMDV表位肽疫苗奠定了基础。此外,通过鉴定小鼠这一FMDV研究中常用动物模型的CTL T细胞表位,也有利于了解细胞毒性淋巴细胞在抗FMDV方面的作用。更多还原

【Abstract】 Epitope vaccine, as a new kind of vaccine, has become one of research hot spots of FMD vaccine. Screening and identification of T cell epitope of FMDV is significant to developing high-efficiency vaccines and further research due to cellular immunity play an important role in immune response of FMDV infection and structural protein VP1 include main antigen sites of FMDV.In this study, The Complete sequence of VP1 of FMDV AF / 72 strains was obtain through cloning and sequencing, and then VP1 protein have high-level expressed in E. coli using the method of codon optimization.The detection of SDS-PAGE and Western Blotting suggest that the molecular weight of expressed VP1 proteins correspond to expected and keep a good immunogenicity. Meanwhile,30 the H-2d Restricted T Cell Epitope of VP1 have been forecast and synthesis using the relative bioinformatics software.The VP1 protein was used for immunizing BALB/c mice and spleen lymphocytes were isolated, and vitro common training stimulus was carried out to potential H-2Dd, H-2Kd and H-2Ld restricted T cell epitope.The H-2Kd restricted T cell epitope pK1 (AYHKGPFTRL) and H-2Dd restricted T cell epitope pD7 (GFIMDRFVKI) were identified through lymphocyte proliferation assay and IFN-γELISPOT experiments.This study provides method and basis through screen and identifies H-2d restricted T cell epitope of structural protein VP1 of foot-and-mouth disease virus type A. in addition, screening and identification of mouse’s CTL epitope contribute to understanding the effect of cytotoxic lymphocytes against FMDV.更多还原