赭曲霉毒素A模拟表位的筛选及免疫学鉴定

SCREENING AND IMMUNOLOGICAL CHARACTERIZATION OF THE MIMICKING EPITOPE OF OCHRATOXIN A

从噬菌体随机七肽库中筛选得到赭曲霉毒素A的模拟表位,并以其替代毒素标品建立赭曲霉毒素A免疫学快速检测方法。以纯化的抗赭曲霉毒素A单克隆抗体(OTA mAb)为配基,亲和筛选融合表达在丝状M13噬菌体次要衣壳蛋白(PⅢ)上的随机七肽库,以ELISA方法鉴定阳性克隆,并以筛选的模拟表位建立赭曲霉毒素A的ELISA检测方法。经过4轮亲和筛选,共得到22株与OTA mAb特异结合的阳性噬菌体克隆,且该结合都能够被OTA标品阻断,DNA测序及多肽核心序列分析后,得到赭曲霉毒素A的模拟表位的主要序列为:MPLWXDL,X为任意氨基酸,以阳性噬菌体克隆建立的竞争ELISA检测方法,线性范围为250～8000pg/ml。基于制备的OTA mAb,利用噬菌体随机七肽库可成功筛选到赭曲霉毒素A的模拟表位,并能够替代OTA标品建立直接竞争模式的免疫学快速检测方法。

To screen the mimicking epitope of Ochratoxin A from Ph. D.-7TM and establish the immunoassay for detecting OTA with it. An anti-Ochratoxin A monoclonal antibody was used as ligand, biopanning was done to screen the mimicking epitope from a Ph.D. -7TM, which was displayed as a fusion protein with the coat protein 11I of filamentous phage M13, the positive clones was identified by ELISA, and established a immunoassay for detecting OTA with the positive phages. After four rounds of panning, 22 positive clones could bind to the OTA mAb and the binding could be blocked by the free ochratoxin A. With the DNA sequencing and peptide core sequence analysis, the amino sequence of the mimicking epitope is MPLWXDL（X is any amino acid residues）. A competitive ELISA immunoassay was established with the positive clones, the linear range of the inhibition curves is between 250pg/ml and 8000pg/ml. Based on the OTA mAb, the phage display technique can be successfully applied to screen the mimicking epitope of Ochratoxin A, the obtain phage can replaced Ochratoxin A to establish the immunoassay.