高致病性禽流感病毒H5N1血凝素蛋白中和抗体及表位的鉴定

Identification of epitopes for neutralizing monoclonal antibodies against highly pathogenic avian influenza H5N1 virus hemagglutinin

目的制备并鉴定中国境内流行的高致病性禽流感病毒H5N1血凝素蛋白(HA)的中和抗体及其表位。方法选取9个HA候选抗原表位序列设计串联表位,利用表面线性表位抗体库(SEAL)专利技术制备单克隆抗体。亲和层析纯化抗体,酶联免疫吸附实验检测亚型及与合成肽结合的剂量依赖性,假病毒微量中和实验筛选中和抗体,免疫印迹法验证与不同毒株HA的结合。结果共制备了23株单抗,其中亚型鉴定10株为IgG型;证明3株IgG3亚型的抗体具有高中和活性,均能与4种不同分枝HA结合;中和抗体识别肽为"LIKKNNAYPT"和"R/KSSFFRNVVW"且具有剂量依赖性。结论鉴定出3株分泌抗中国境内流行H5N1 HA的中和抗体,其对应的HA抗原表位为中和表位,可作为H5N1疫苗研制的候选表位。

Objective To generate and characterize neutralizing monoclonal antibodies against highly pathogenic avian influenza （HPAI） H5N1 strains and corresponding epitopes.Methods Nine candidate peptides of HAs were designed to generate monoclonal antibodies using a patent technology of SEAL （surface epitope antibody library）.Ascites was purified by affinity chromatography.Neutralizing monoclonal antibodies were characterized by ELISA,Western blot and micro-neutralization assays.Results Twenty-three hybridomas clones anti-HA epitope monoclonal antibodies were obtained,in which 3 IgG3 subtype clones showed efficient neutralizing activity to H5N1 pseudotype virus and recognized corresponding epitopes ＂ LIKKNNAYPT ＂ and ＂ R/KSSFFRNVVW ＂ in a dose dependent manner.These 3 mAbs recognized rHA from 4 different branches of H5N1 demonstrated by immunoblot.Conclusions 3 high levels of neutralizing monoclonal antibodies were characterized.Two important neutralizing epitopes of HA were identified which might serve as a strong potential candidate for an effective vaccine to protect from highly pathogenic avian influenza H5N1 virus in China.