随机肽库筛选赭曲霉毒素A模拟表位及其应用
Selecting mimotope of ochratoxin A from phage random peptide library and its application
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摘要:目的 从噬菌体随机肽库中筛选模拟赭曲霉毒素A表位的噬菌体粒子, 并以其替代毒素建立免疫学检测方法。方法 以抗赭曲霉毒素A的单克隆抗体为配基, 免疫亲和筛选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机七肽库, 以ELISA方法鉴定阳性克隆, 同时进行DNA测序确定插入七肽的氨基酸序列。结果 经过4轮淘选, 共筛选到11株能与该抗体特异性结合的阳性克隆, 且该结合能被赭曲霉毒素A阴断, 模拟表位的共有序列为: 异亮氨酸—精氨酸—脯氨酸—蛋氨酸—缬氨酸—X—X, X为任意氨基酸。以其中阻亲和力最强的克隆(P2)建立了竞争ELISA检测方法, 线性范围为200~8000pg/ml, 检测下限为150pg/ml。结论 噬菌体展示技术可成功筛选到赭曲霉毒素A模拟表位, 筛选到的噬菌体粒子可作为毒素的替代品建立免疫学分析方法。Abstract:Objective To screen phage particles capable of mimicking ochratoxin A and to establish immunoassay for ochratoxin A with it.Methods An anti-ochratoxin A monoclonal antibody was used as ligand.Biopanning was done to screen the mimotope from a phage random 7 peptide library.This library was displayed as a fusion protein with the coat protein Ⅲ of filamentous phage M13.The positive clones were identified by ELISA, and the inserted amino sequences were deduced by DNA sequencing.Results After four rounds of panning, 11 positive clones could bind to the antibody, and the binding could be blocked by free ochratoxin A.The common amino sequence of the mimicking epitope was IRPMVXX.A competitive ELISA immunoassay was established with clone P2;the linear range of the inhibition curves was between 200 pg/ml and 8 000 pg/ml; the detecting limitation was 150 pg/ml.Conclusion The phage display technique could be successfully applied to screen the mimotope of ochratoxin A.The acquired phages might be used as the surrogate of the mycotoxin to establish the immunoassay.
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