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(M2; e)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M2 e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M2; e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)-M2 e及M2; e-(CRM197-N190)主要以类似M2 e天然构象的四聚体形式存在;融合蛋白可与多株流感M2; e特异性单抗以及CRM197特异性单抗反应,M2; e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M2; e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 M2 eprotein is a potential candidate for development of a; universal vaccine against influenza viruses.In this work,M2 ewas 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 M2 e; protein.The fusion protein of M2 efused to the N-terminus of; CRM197,CRM197-N389,and CRM197- N190 showed excellent reactivity to the; characterized monoclonal antibodies O19 and L18 as compared with M2; efused 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 M2; ewas enhanced significantly when being fused to CRM197-N190 than to; GST.These findings provide some clues for the development of a M2; e-based,broad-spectrum influenza vaccine.国家自然科学基
