流感病毒M2e与白喉毒素无毒突变体CRM197融合蛋白的抗原性和免疫原性分析  

Antigenicity and Immunogenicity of Influenza M2e Protein Fused with Intramolecular Adjuvant CRM197

作　　者：李琼[1] 王凯航[1] 周立志[2] 陈婷婷[1] 张晓[2] 陈毅歆[1,2] 顾颖[1,2] 夏宁邵[1,2] 李少伟[1,2] 

机构地区：[1]厦门大学生命科学学院,国家传染病诊断试剂与疫苗工程技术中心,厦门361102 [2]厦门大学公共卫生学院,分子疫苗学和分子诊断学国家重点实验室,厦门361102

出　　处：《病毒学报》2017年第4期483-493,共11页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31670934),题目:基于广谱中和单抗的通用型流感疫苗设计及其结构基础研究~~

摘　　要：流感病毒严重威胁人类健康且流行株不断变化,传统疫苗对流感预防具有局限性。流感病毒M2蛋白胞外区(M2e)氨基酸序列高度保守,可诱导产生特异性抗体,是通用流感疫苗研究的主要靶标之一。白喉毒素突变体(CRM197)是一种理想的蛋白载体,可增强小分子抗原的免疫原性,商业化的肺炎球菌疫苗采用CRM197结合形式提高多糖分子的免疫原性。本实验通过重组蛋白融合的方式,将M2e与CRM197(aa 1~535)、CRM197-N190(aa 1~190)、CRM197-N389(aa 1~389)的C端或N端进行融合表达,并考察融合蛋白的反应活性以及免疫原性。融合蛋白中的M2e和CRM197均具有反应活性;超速离心分析和四聚体特异构象单抗反应均表明融合蛋白(CRM197-N190)-M2e及M2e-(CRM197-N190)主要以类似M2e天然构象的四聚体形式存在;融合蛋白可与多株流感M2e特异性单抗以及CRM197特异性单抗反应,M2e融合至CRM197N末端形成的重组蛋白的反应活性高于C端融合蛋白;相比融合GST,与CRM197-N190的融合显著增强了M2e蛋白的免疫原性,为流感通用疫苗研究提供了新思路。New strategies are needed urgently to develop broad-spectrum vaccines against the influenza virus and to reduce the risk of pandemics.The conserved M2e protein is a potential candidate for development of a universal vaccine against influenza viruses.In this work,M2e was fused to the N-or C-terminus of CRM197（aa1~535）,CRM197-N389（aa1~389）and CRM197-N190（aa1~190）by gene engineering.Data from SDS-PAGE and western blotting showed that these fusion proteins formed multimers,and that the monomer and dimer bands were the most prominent.Moreover,AUC data demonstrated that the tetramers of some fusion proteins were present in solutions that might reflect the native conformation of the M2e protein.The fusion protein of M2e fused to the N-terminus of CRM197,CRM197-N389,and CRM197-N190 showed excellent reactivity to the characterized monoclonal antibodies O19 and L18as compared with M2e fused to the C-terminus of CRM197,CRM197-N389,and CRM197-N190.Furthermore,the immunogenicity of these fusion proteins was evaluated in mice.Results demonstrated that the immunogenicity of M2e was enhanced significantly when being fused to CRM197-N190 than to GST.These findings provide some clues for the development of a M2e-based,broad-spectrum influenza vaccine.

关 键 词：流感病毒 M2e蛋白 CRM197 抗原性 免疫原性 

分 类 号：R373.1[医药卫生—病原生物学]