利用噬菌体随机肽库筛选传染性法氏囊病病毒VP2模拟表位  

Screening of Mimic Epitopes for Infectious Bursal Disease Virus VP2 by Phage Display Random Peptide Library

作　　者：王小娥[1,2] 杨春晓[1,3] 朱彩霞[1] 孙寅剑[1] 崔金生[1] 肖海君[1] 朱瑞良[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018 [2]瑞普(天津)生物药业有限公司,天津300300 [3]济宁市畜牧兽医局,济宁272037

出　　处：《农业生物技术学报》2010年第3期573-579,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(30571371);山东省博士后科研项目择优基金;山东省科技攻关项目(2008GG10009023);高等学校博士学科点专项科研基金(20060434011);国家人事部博士后基金;山东省优秀中青年科学家科研奖励基金(03BS140)共同资助

摘　　要：用纯化的4株传染性法氏囊病病毒(IBDV)VP2单克隆抗体(1B5,5D1,2H11和I4-4-3)筛选噬菌体随机12肽库,对40个克隆(10个单克隆噬菌斑×4株单克隆抗体)进行ELISA验证,获得32个阳性克隆,选20个阳性克隆(5个单克隆噬菌斑×4株单克隆抗体)进行DNA序列分析,确定了4个优势12肽为不同传染性法氏囊病病毒(IBDV)抗原表位。这4个12肽在一级结构上没有3个以上连续氨基酸与GenBank中传染性法氏囊病病毒VP2的氨基酸序列相同之处,与单抗的结合可被VP2蛋白有效地抑制,推测可能是构象依赖性表位。将4个模拟表位串联表达后获得目的蛋白,经SDS-PAGE分析,目的蛋白占菌体总蛋白的20%,分子量30kD。用多克隆抗体对目的蛋白进行免疫印迹分析,结果表明目的蛋白具有特异性和反应原性。试验结果提示:筛选的是传染性法氏囊病病毒(IBDV)VP2单抗的模拟表位。The 12-mer phage random peptide library was screened with four purified mAbs against infectious bursal disease virus （IBDV） VP2 （1B5, 5D1, 2H11 and I4-4-3） and 40 clones were obtained, among which, 32 positive clones were identified by ELISA.Twenty positive clones for DNA sequence analysis were chosen and four ascendant 12-peptides were determined as different epitopes of infectious bursal disease virus （IBDV） VP2. The four ascendant 12-peptides shared no more than three continuous amino acid residues similar with the sequences of infectious bursal disease virus （IBDV） VP2 registered in GenBank, and their conjunction with mAbs against infectious bursal disease virus （IBDV） VP2 could be inhibited by VP2 protein,which indicated that the epitopes were conformation-dependent. When the four mimicking epitopes were expressed tandem, the interest protein was obtained. The results from SDS-PAGE analysis showed that the proportion of the interest protein was 20% of the total bacterial proteins and the molecular weight was 30 kD. The the interest protein showed specificity and reactionogenicity by use of immunoblotting test with polyclonal antibodies. Imagine： Four conformation-dependent mimic epitopes of mAbs against infectious bursal disease virus （IBDV） VP2 were obtained by phage display technique.

关 键 词：模拟表位 噬菌体随机12肽库 噬菌体展示技术 

分 类 号：S852.659.4[农业科学—基础兽医学]