应用噬菌体随机肽库技术筛选SARS-CoVS蛋白模拟表位  

Screening and identification of mimotope of the SARS-CoV spike protein by phage peptide library

作　　者：张小萍[1] 王靖雪[1] 魏静[1] 万瑛[1] 吴玉章[1] 

机构地区：[1]第三军医大学全军免疫学研究所,重庆400038

出　　处：《免疫学杂志》2005年第6期442-444,452,共4页Immunological Journal

基　　金：国家自然科学基金重大项目(30490240);国家杰出青年科学基金(30325020)资助

摘　　要：目的筛选SARS病毒(Severe acutere spiratorysyndromeas sociated coronavirus,SARSCoV)结构蛋白S(Spike)特异性的模拟表位,为抗SARS疫苗研究提供基础。方法以抗SARS病毒S蛋白的单克隆抗体为固相筛选分子,对人工合成的噬菌体随机12肽库进行5轮“吸附洗脱扩增”的筛选,随机挑选30个克隆,经噬菌体酶联免疫吸附(ELISA)法和交叉反应实验鉴定阳性克隆,再进行DNA序列分析和竞争抑制结合实验,以确定SARS病毒S蛋白的模拟表位。结果经噬菌体富集后,从随机挑选的30个克隆中得到了29个编码8种12肽的阳性克隆,8条肽与S蛋白的320～350氨基酸序列有较高同源性,确定YF、W、E和K氨基酸残基为模拟表位的骨架结构。结论用噬菌体12肽库成功筛选到了SARS病毒S蛋白的模拟表位,为基于S蛋白的肽疫苗研制提供了基础。Objective To screen the mimotopes of SARS-CoV spike protein by using monoclonal antibody and phage peptide library. Methods By using SARS-CoV spike protein specific monoclonal antibody as selective molecule, a 12-mer phage peptide library was biopanned and positive clones were selected by ELISA,competition assay, and DNA sequencing. Results There were approximately 20000 times of enrichment about the titer of bound phages after five rounds biopanning procedures. Twenty-nine positive clones encoding 8 different 12-mer peptides were selected. These peptides were highly homologous with 320 - 350 amino acid sequence of SARS-CoV S protein, and most of the phage clones had the motifs of F/Y（X）WE（X）K. Conclusion SARS-CoV S protein mimotope is obtained by phage peptide library screening, which provides bases for anti-SARS-CoV peptide vaccine development.

关 键 词：SARS病毒 S蛋白 模拟表位 噬菌体肽库 

分 类 号：R392.1[医药卫生—免疫学]