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

doi:10.7668/hbnxb.2015.01.023

摘要/Abstract

摘要： 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200～213aa)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200～213aa),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。

关键词: 兔出血症病毒, 病毒样颗粒, 口蹄疫病毒, B细胞表位, 载体

Abstract: 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 306th and 307th aa of capsid protein of RHDV.The fused genes were cloned into the donor vector pFastBac^TMHTA 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 effectively 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 electron microscopy analysis.Furthermore, all chimeric VLPs were able to induce strong VP60-specific antibodies responses.In addition, there were significant differences of peptide-specific IgG antibodies induced by chimeric proteins, 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.

Key words: RHDV, Virus-like particles, FMDV, B-cell epitope, Vector

中图分类号:

盛蓉, 宋艳华, 胡波, 范志宇, 姜平, 薛家宾, 魏后军, 王芳. 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[J]. 华北农学报, 2015, 30(1): 142-149. doi: 10.7668/hbnxb.2015.01.023.

SHENG Rong, SONG Yan-hua, HU Bo, FAN Zhi-yu, JIANG Ping, XUE Jia-bin, WEI Hou-jun, WANG Fang. Study on the Expression and Immunogenicity of Chimeric Rabbit Hemorrhagic Disease Virus-like Particles as Carriers for B-cell Epitope of Foot-and-mouth Disease Virus[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(1): 142-149. doi: 10.7668/hbnxb.2015.01.023.

http://www.hbnxb.net/CN/Y2015/V30/I1/142

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