抗口蹄疫病毒分泌型IgA抗体的构建表达及特性分析

Construction, expression, and characterization of secretory IgA antibody against foot-and-mouth disease virus

摘要:

目的 制备抗口蹄疫病毒(foot-and-mouth disease virus, FMDV)分泌型IgA (secretory IgA, sIgA)抗体，为口蹄疫(foot-and-mouth disease, FMD)的综合防控提供新思路和前期基础。方法 以实验室前期筛选的一株抗O型和A型FMDV的IgG型中和抗体POA-8为模板，用猪IgA重链的恒定区基因(经密码子优化)替换IgG重链的相应区域，构建抗FMDV IgA抗体的重链真核表达质粒；同时分别构建含猪源J链和分泌片(secretory component, SC)的真核表达质粒，以及同时含IgA抗体的重链、轻链和J链基因的真核表达质粒。采用4质粒(重、轻、J和SC)或2质粒(重+轻+J和SC)分别瞬时共转染CHO-S悬浮细胞，进行抗FMDV sIgA抗体的组装表达。表达的sIgA抗体经纯化后，通过SDS-PAGE和Western blotting进行鉴定，并利用间接ELISA和微量病毒中和实验评价sIgA抗体与FMDV的结合活性和中和活性。结果 4质粒或2质粒共转染CHO-S细胞后均能成功组装表达抗FMDV的sIgA抗体，且sIgA抗体对O型和A型FMDV的结合能力及中和能力均强于IgG抗体。结论 本研究成功构建了抗FMDV sIgA抗体的表达系统，为未来FMD黏膜疫苗和抗病毒药物的开发奠定了基础。

Abstract:

Objective To prepare secretory IgA (sIgA) antibody against foot-and-mouth disease virus (FMDV) and provide a new idea and basis for the comprehensive prevention and control of foot-and-mouth disease (FMD).Methods We constructed an eukaryotic expression plasmid harboring the heavy chain of IgA antibody by replacing the constant region gene of heavy chain of IgG with the codon-optimized corresponding region of IgA based on POA-8, a previously screened IgG neutralizing antibody capable of neutralizing both type O and type A FMDV. At the same time, the eukaryotic expression plasmids harboring the J chain and secretory component (SC) of pigs as well as the eukaryotic expression plasmid containing heavy chain, light chain, and J chain genes were constructed respectively. CHO-S cells were transiently co-transfected with 4 plasmids (heavy, light, J, and SC) or 2 plasmids (heavy+light+J and SC) for assembling and expression of sIgA antibody. sIgA antibody was purified and identified by SDS-PAGE and Western blotting. The binding activity and neutralizing activity of sIgA antibody against FMDV were evaluated by indirect ELISA and virus neutralization assay.Results sIgA antibody against FMDV could be successfully assembled and expressed after transfection of CHO-S cells with 4 or 2 plasmids. The binding and neutralizing abilities of sIgA against type O and type A FMDV were stronger than those of IgG.Conclusion This study established an efficient expression system for sIgA against FMDV, laying a foundation for the development of mucosal vaccines and antiviral drugs against FMDV in the future.