Objective:To screen the neutralizing epitope of enterovirus 71 (EV71) and determine the specific minimum amino acid sequence that triggers immunity for providing a theoretical basis for the development of synthetic peptide vaccines.Methods:EV71 neutralizing antibody-specific binding clones were panned and sequenced using a phage display random 12-peptide library to obtain the key sequences of neutralizing epitopes. A series of peptides containing the key sequences with N-terminal acetylation (AC) and C-terminal linking to Keyhole limpet hemocyanin (KLH) were synthesized. Serum samples were collected after immunizing mice with the modified peptides. Then the immunogenicity of the peptides and the neutralizing activity of serum samples were analyzed by Western blot, ELISA and neutralization test.Results:After three rounds of panning, cloning and sequencing, KQEKDL was identified as the key motif. The serum samples collected from the mice immunized with the modified series of peptides containing key motifs had different degrees of binding ability to EV71 and VP1 protein. The serum samples of mice immunized the synthetic peptide containing only the minimum key motif (AC-KQEKDL-KLH) had the strongest response to the other three peptides and EV71 and the highest neutralizing titer.Conclusions:The EV71 neutralizing epitope was successfully screened using the phage display random peptide library. The key motif of KQEKDL might be the specific minimum amino acid sequence that triggered the immune system. This study provides a theoretical basis for better understanding the immune response mechanism, evaluating the immunogenicity of the antigens and further research and development of polypeptide vaccines.

柯萨奇病毒A16(CV-A16)是小RNA 病毒科肠道病毒属的成员,是引起婴幼儿手足口病(H FM D )的常见病原体之一[1].CV-A16是含有约7 .4 kb基因组的正义单链RN A病毒,其基因组包含5'端非翻译区(5'U T R )、结构蛋白编码区P1、非结构蛋白编码区P2/P3及3'非翻译区(3'U T R ).P1编码4种结构蛋白(V P1、V P2、V P3、V P4 ) ,P2和P3分别编码7种非结构蛋白(2A、2B、2C、3A、3B、3C、3D )[2].随着研究的不断深入,在分子流行病学方面,基于V P1基因的完整序列,CV-A16可以在系统发育上分为A、B、C和D 4个基因组[3-6 ].基因组B可以进一步分为B1～B4这4 个基因亚型,其中B1 基因亚型还可以细分为B1a～B1d[3 ,7].在基础研究方面,利用反向遗传学技术构建感染性克隆[8 ] ,能够在体外对病毒基因组进行突变、缺失、嵌合等操作后,获得表型、性状发生变化的拯救病毒,为研究其基因的结构与功能、病毒复制机制、致病机制及筛选候选疫苗提供了崭新的途径.在疾病预防控制方面,单价肠道病毒71 型(EV-A71)疫苗无法预防其他病原体引起的 HFMD及其他相关疾病,应研发多价HFMD 疫苗[9] ,而EV-A71和CV-A16交替或共同流行可引发世界多个国家或地区HFMD暴发.本研究从CV-A16的分离、鉴定、系统进化分析、检测方法建立和全基因组克隆构建等方面进行了一系列研究,以期为C V-A 16 病毒的分子流行病学和基础研究提供有利的工具.