从噬菌体随机肽库筛选庚型肝炎病毒E2抗原表位  

Bio-panning of HGV E2 epitope from a phage-displayed random peptide library

作　　者：柯屾 戚中田[1] 施明[1] 赵平[1] 王建安[2] 姚志建 沈倍奋[2] 

机构地区：[1]第二军医大学基础医学部微生物学教研室,上海200433 [2]军事医学科学院基础医学研究所 [3]北方基因研究中心

出　　处：《第二军医大学学报》2000年第9期838-841,共4页Academic Journal of Second Military Medical University

基　　金：国家"8 63"计划资助!项目 ( 10 2 -0 9-0 1-0 2 ) ;国家自然科学基金!重点资助项目 ( 3 983 0 3 3 0 )

摘　　要：目的 :筛选能识别庚型肝炎病毒 (HGV) E2区的单克隆抗体和有竞争抑制活性的多肽。方法 :利用抗 HGV E2区的3株单克隆抗体 M6 ,M13,M30作为筛选配基 ,对构象限制性的随机环 9肽库进行亲和筛选 ,并对阳性噬菌体克隆进行功能鉴定。结果 :证实亲和筛选具有良好的富集效果后 ,随机挑出 16个噬菌体克隆进行交叉结合实验 ,有 14个克隆与 M6抗体有较强的特异结合 ;其中 10个克隆在竞争抑制实验中抑制率大于 5 0 %。 DNA测序表明 9个克隆具有氨基酸核心序列 GYAPL S,该序列与 HGV E2区第 30 6～ 311位氨基酸有较好的同源性。用合成肽与核心序列相似的噬菌体克隆 P9GC5和 P9GC11进行HGV E2抗原抗体实验 ,显示它们的 IC5 0 分别为 4.5和 7.2 nmol/ L。结论 :从噬菌体随机肽库中筛选到能与 HGV E2抗体特异性结合的 HGV抗原表位 ,并确定与合成了具有活性的核心多肽序列。Objective: To study Bio panning of hepatitis G virus(HGV) E2 with monoclonal antibody(mAb) from a nonapeptide library. Methods: Three mAb(M6, M13 and M30) against E2 protein of HGV were used for bio panning of a phage displayed random nonapeptide library. Results: After 3 rounds of effective screening, sixteen positive phage clones were selected for binding and competitive inhibition tests. Fourteen out of the 16 clones could specifically react with mAb M6. The inhibition rates of 10 clones out of 14 were over 50%. From the deduced insert sequence in the phage clone, the core sequence of amino acid GYAPLS was found in 9 clones. This core sequence was equivalent to amino acid sequence of 306 311 of HGV E2.The phage clone P9GC5 and P9GC11 containing motif sequence competitively inhibited the binding of HGV E2 with the mAb, with 4.5 and 7.2 nmol/L of IC 50 respectively. Conclusion: Our finding indicates that GYAPLS motif is the mimic of HGV E2 epitope that can be recognized by HGV mAb M6. [

关 键 词：噬菌体随机肽库 E2蛋白 抗原表位 庚型肝炎 HGV 

分 类 号：R512.63[医药卫生—内科学] Q785[医药卫生—临床医学]