携带口蹄疫病毒B细胞表位的兔出血症病毒样颗粒的表达及其免疫原性研究

摘要

To explore the capacity of antigen presentation of the foreign B-cell epitopes by virus-like particles (VLPs) of rabbit hemorrhagic (RHDV),the VLPs of RHDV displaying B-cell epitope of foot-and-mouth disease virus (FMDV) were constructed.The sequences of a FMDV B-cell epitope were fused to the N-terminal,C-terminal and inserted between 306 th and 307 th aa of capsid protein of RHDV .The fused genes were cloned into the donor vector pFastBacTM HTA and three recombinant baculoviruses (rAc-VP60-2F,rAc-VP60-306F,rAc-VP60-578F) were constructed using Bac-to-Bac baculovirus expression system .These recombinant proteins were expressed effec-tively in insect cells as confirmed by IFA ,SDS-PAGE and Western Blot .The immunogenicity of all chimeric VLPs was examined in mice .The results indicated the recombinant proteins could react with both anti-VP60 monoclonal antibody and anti-FMDV polyclonal antibody .And all recombinant proteins could self-assemble into VLPs by elec-tron microscopy analysis .Furthermore, all chimeric VLPs were able to induce strong VP 60-specific antibodies re-sponses .In addition ,there were significant differences of peptide-specific IgG antibodies induced by chimeric pro-teins,compared to that of VP60 group.The significant levels of serum antibodies against B-cell epitopes of FMDV demonstrated the feasibility of RHDV-VLP serving as a presentation carrier for foreign B-cell epitopes .%为了研究RHDV病毒样粒子作为载体提呈外源B细胞表位的能力，以兔出血症病毒（ RHDV ）衣壳蛋白VP60为载体，构建携带FMDV B细胞表位（ FMDV VP1 B细胞表位200～213 aa ）的VP60嵌合蛋白，研究外源基因对VP60蛋白的表达、病毒样颗粒（ VLPs）的装配及免疫原性的影响。分别在RHDV衣壳蛋白的C端、N端、306和307位氨基酸之间插入外源B细胞表位（ FMDV VP1 B细胞表位200～213 aa ），得到嵌合VP60基因。利用杆状病毒表达系统表达嵌合蛋白，分别命名为VP60-2F、VP60-306F和VP60-578F。经IFA、SDS-PAGE和Western Blot方法鉴定嵌合蛋白的表达，通过电镜观察嵌合蛋白自聚为病毒样颗粒的能力，通过动物试验研究其免疫原性。结果显示，嵌合VP60蛋白在杆状病毒表达系统中得到高效表达，可自聚形成病毒样颗粒，免疫小鼠后可以产生针对VP60和B细胞表位特异的免疫应答。该研究为嵌合VP60 VLPs的形成及结构功能的研究奠定基础，同时为RHDV-VLPs作为外源B细胞表位展示载体的可行性提供依据。