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2025年4月10日 星期四
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利用噬菌体随机肽库筛选IBDV-VP2模拟表位*
王小娥
Screening of mimic epitopes for IBDV-VP2 by Phage Display Random Peptide Library
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摘要 目的:获得具有中和作用的IBDV-VP2单抗的模拟表位,为进一步研究其对法氏囊病的免疫保护作用奠定基础。方法:用纯化的4株IBDV-VP2单克隆抗体(1B5、5D1、2H11和I-4-4-3)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IBDV-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r4EPIS蛋白,经SDS-PAGE分析,r4EPIS占菌体总蛋白的20%,分子量30kDa。用多克隆抗体对r4EPIS进行免疫印迹分析,结果表明r4EPIS具有特异性和反应原性。结论:筛选的是IBDV-VP2单抗的模拟表位。
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王小娥
关键词 模拟表位噬菌体随机12肽库噬菌体展示技术    
Abstract:Purpose: To obtain mimicking epitopes of mAbs against infectious bursal disease virus (IBDV) VP2 using phage display technique, so as to offer basis for further study of their immuno-protection against infectious bursal disease (IBD). Methods: The 12-mer phage random peptide library was screened with four purified mAbs against IBDV-VP2(1B5、5D1、2H11 and I-4-4-3 ) and 40 clones were obtained, among which, 32 positive clones were identified by ELISA.We chosen 20 positive clones for DNA sequence analysis and four ascendant 12-peptides were determined as different epitopes of IBDV-VP2. The four ascendant 12-peptides shared no more than three continuous amino acid residues similar with the sequences of IBDV-VP2 registered in GenBank, and their conjunction with mAbs against IBDV-VP2 can be inhibited by VP2 protein,which indicated that the epitopes were conformation-dependent. When the four mimicking epitopes were expressed tandem, protein r4EPIS was obtained. The results from SDS-PAGE analysis showed that the proportion of r4EPIS was 20% of the total bacterial proteins and the molecular weight was 30 kDa. The r4EPIS showed specificity and reactionogenicity by use of immunoblotting test with polyclonal antibodies. Conclusion: Four conformation-dependent mimic epitopes of mAbs against IBDV-VP2 were obtained by phage display technique.
Key wordsMimic Epitope    12-mer phage random peptide library    phage display technique
收稿日期: 2009-07-17     
通讯作者: 王小娥   
引用本文:   
王小娥. 利用噬菌体随机肽库筛选IBDV-VP2模拟表位*[J]. , 2010, 18(3): 573-579.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2010/V18/I3/573
 
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