A型口蹄疫病毒衣壳蛋白主要抗原表位区的原核表达及免疫效果分析

Prokaryotic expression and immune effect of major antigenic epitope regions of type A foot-and-mouth disease virus capsid protein

为了探索更有效的A型口蹄疫(FMD)表位疫苗,根据已鉴定的A型口蹄疫病毒(FMDV)主要抗原位点,结合基因序列比对分析,选择了A型FMDV流行毒株的主要抗原位点区进行表位蛋白分子的设计表达。所用表位序列包括A/QH/CHA/2013的VP2 70~81位、118~140位,VP1 G-H环131~157位、C末端188~212位,A/WH/CHA/09 VP1 43~48位、VP3 55~72位,以及非结构蛋白3A中的T细胞表位和通用性T细胞表位,构建免疫原蛋白分子命名为ANX2和ANX3,其中ANX2采用了表位序列反向串联的方式进行设计,用大肠杆菌表达免疫原基因,纯化表位蛋白免疫小鼠和猪,分析特异性体液免疫和细胞免疫应答水平,并对免疫猪进行了攻毒保护试验,评价表位疫苗的保护效果。结果显示,ANX2、ANX3重组蛋白以包涵体表达为主,蛋白大小约为41 ku和39 ku,均能与A型FMDV阳性血清发生特异性反应;免疫小鼠和猪后都能产生抗A型FMDV特异性抗体;用表位蛋白刺激免疫小鼠脾淋巴细胞,细胞因子IFN-γ和IL-4均有明显的升高;攻毒后对猪有一定的保护作用,ANX2的免疫效果略优于ANX3,证明表位序列反向串联更有利于抗原表位的展示,从而增强其免疫效果。本研究为A型FMDV表位疫苗的研究积累了经验。

In order to develop a more effective epitope vaccine,two epitope-based immunogens were designed referring to the previously-reported antigenic sites of foot-and-mouth disease virus(FMDV)type A field strains.The epitopes selected in this study included:amino acids sites 70-81 and 118-140 of VP2, 131-157 and 188-212 of VP1 from A/QH/CHA/2013 strain,amino acids sites 43-48 of VP1 and55-72 of VP3 from A/WH/CHA/09 strain,one T cell epitope in 3 A and a universal T cell epitope.The two designed epitopes were named as ANX2 and ANX3,respectively.ANX2 was designed by reversing the sequences of some B cell epitopes compared with ANX3.Epitope proteins were expressed in E.coli and purified for preparation of the vaccines.After vaccination to mice and pigs,the levels of specific humoral and cellular immune responses were analyzed.The pigs were challenged with the epidemic strains to evaluate the protective efficiency of the vaccines on the 28 th day after immunization.The results showed that two epitope proteins were mainly expressed in inclusion bodies,and the molecular weight of two proteins were about 41 ku and 39 ku respectively.Both of two proteins can specifically react with positive serum of FMDV type A by Western-blot.Mice and pigs vaccinated with epitope-based vaccines produced FMDV type A specific antibody and the responses level of IFN-γand IL-4 cytokines were increased after in vitro stimulation of lymphocytes in mice with epitope proteins. Partial protective effects were observed in pigs vaccinated with two vaccines after challenge with virulent type A virus,and ANX2 showed better protective effect than ANX3,which indicating that the reverse sequence construction displayed the epitope more like natural conformation.The study accumulates some useful data for development of the epitope-based vaccine against FMDV type A.