Development of a multi-mimotope peptide as a vaccine immunogen for infectious bursal disease virus
Development of a multi-mimotope peptide as a vaccine immunogen for infectious bursal disease virus
复制标题
DOI：
10.1016/j.vaccine.2007.03.018
复制
发表时间：
2007-05-30
期刊：
影响因子：
5.5
通讯作者：
Lu Cheng-ping
中科院分区：
文献类型：
--
作者：
Wang Yong-shan;Fan Hong-jie;Lu Cheng-ping
为探索传染性法氏囊病病毒（IBDV）模拟表位疫苗的研究方法，制备了5株IBDV特异性单克隆抗体，并利用噬菌体展示12肽库筛选了5株单克隆抗体的结合肽。经3轮生物淘选，筛选出12株单抗，用夹心法和竞争抑制ELISA法检测其对IBDV的特异性。测定了7株单克隆抗体的核苷酸序列，并推导了其氨基酸序列。确定了5个代表性的mimotopes序列。人工合成了5个表位（5 epis，5 epitopes）的人工基因FI-F7-B34-2B-2G 8，并将其克隆到原核表达质粒pET 28 b中。在大肠杆菌中表达了重组蛋白r5 EPIS，SDS-PAGE显示其分子量约为10 kDa。Western blot检测r5 EPIS蛋白与抗IBDV单克隆抗体和多克隆抗体反应。r5 EPIS蛋白免疫SPF鸡的研究（用弗氏佐剂，在第0天和第14天每次注射50 μ g）诱发高水平的抗体（在第21天，ELISA检测为12，800/病毒中和试验为1600），并且保护100%的鸡抵抗200 ELD 50的IBDV GX 8/99毒株的攻击，这分别与，佐剂组和未治疗组的存活率分别为13.3%和6.6%。r5 EPIS是一种新型的IBDV亚单位疫苗候选蛋白。(C)2007爱思唯尔有限公司保留所有权利。
To explore the mimotope vaccine approach against infectious bursal disease virus (IBDV), five IBDV-specific monoclonal antibodies (mAbs) were prepared and their binding peptides were screened against a phage-displayed 12-mer peptide library. After three rounds of biopanning, 12 phages were selected for each mAbs and their specificity to IBDV was verified by sandwich and competitive inhibition ELISAs. Seven phages per mAb were sequenced and their amino acid sequences were deduced. The five representative sequences of mimotopes corresponding mAbs were determined. An artificial gene, designated 5epis (5 epitopes) and consisting of the five mimotopes arranged in tandem (FI-F7-B34-2B-2G8) with four GGGS spacers, was chemically synthesized and cloned into a prokaryotic expression plasmid pET28b. The protein, designated r5EPIS, was efficiently expressed in Escherichia coli and showed a size of 10kDa in SDS-PAGE. The r5EPIS protein reacted with anti-IBDV mAbs and polyclonal antibodies in Western blot immunoassays. Immunization of SPF chickens with r5EPIS protein (with Freund adjuvant, 50 jig per injection on day 0 and 14) evoked high levels of antibody (12,800 by ELISA/1600 by virus neutralizing assay at day 21) and protected 100% of the chickens against a challenge of 200 ELD50 of IBDV GX8/99 strain, which sharply contrasted with the, respectively, 13.3% and 6.6% survival rate in the adjuvant group and the untreated group. The multi-munotope protein r5EPIS promises to be a novel subunit vaccine candidate for IBDV. (C) 2007 Elsevier Ltd. All rights reserved.