RHDV VLPs对口蹄疫病毒B细胞表位的展示效果

摘要

To explore the capacity of antigen presentation of the foreign B cell epitopes by virus⁃like particles ( VLPs) of rabbit hemorrhagic disease virus ( RHDV) , the sequences of multi⁃copy B⁃cell epitopes of foot⁃and⁃mouth dis⁃ease virus (FMDV) [FMDV VP1 GS⁃(200⁃213 aa)⁃GS⁃(141⁃160 aa)] were fused to N⁃terminal and C⁃terminal and in⁃serted between 306th and 307th amino acid of capsid protein of RHDV. The fused genes were cloned into the donor vector pFastBacTM HTA and three recombinant baculoviruses ( rAc⁃VP60⁃2FB, rAc⁃VP60⁃306FB, rAc⁃VP60⁃578FB ) were obtained using Bac⁃to⁃Bac baculovirus expression system. These chimeric proteins were expressed effectively in insect cells as confirmed by IFA, SDS⁃PAGE and Western blot. The immunogenicity of all chimeric proteins was eval⁃uated in mice. The results showed that the chimeric proteins could react with both anti⁃VP60 monoclonal anti⁃body and anti⁃FMDV polyclonal antibody. And all recom⁃binant proteins could self⁃assemble into VLPs under electron microscopy, and were able to induce VP60⁃specific antibodies responses. The peptide⁃specific antibody maintained a high level when the inserted foreign fragments were up to 126 bp. The length of the foreign genes carried by VP60⁃VLPs was extended in this study, which demonstrated the feasibility of VP60⁃VLPs serving as a presentation carrier for foreign B⁃cell epitope.%为研究兔出血症病毒（ RHDV）病毒样颗粒（ VLPs）携带外源表位的能力及其免疫原性，以兔出血症病毒（ RHDV）衣壳蛋白VP60为载体，分别在RHDV VP60蛋白质的C端、N端和306位插入FMDV VP1双串联B细胞表位[GS⁃（200～213 aa）⁃GS⁃（141～160 aa）]，得到嵌合蛋白质，分别命名为 VP60⁃2FB、VP60⁃306FB 和 VP60⁃578FB。经IFA、SDS⁃PAGE和Western blot鉴定，嵌合VP60蛋白质在杆状病毒表达系统中均得到有效表达；通过电镜观察发现嵌合蛋白质可自聚成病毒样颗粒。将嵌合蛋白质免疫小鼠，检测产生的免疫应答情况，结果显示，嵌合蛋白质可以产生针对VP60特异的免疫应答，且在插入长度为42 aa外源氨基酸片段的情况下，嵌合蛋白质免疫的小鼠仍能诱导产生较高水平的针对外源表位的特异性应答。在进一步扩展了载体的容纳性后，序列的增加不影响VLPs的形成以及对外源表位的递呈效果，说明VP60⁃VLPs作为外源B细胞表位展示载体是可行性的。