脱氧雪腐镰刀菌烯醇的环七肽模拟表位筛选

余鹃; 贾彦琼; 骆敏儿; 王宏

doi:10.13386/j.issn1002-0306.2017.24.026

脱氧雪腐镰刀菌烯醇的环七肽模拟表位筛选

Screening of the mimic epitopes of deoxynivalenol from cyclo-heptapeptide library

摘要

摘要: 利用噬菌体随机环七肽库筛选脱氧雪腐镰刀菌烯醇（Deoxynivalenol,DON）模拟表位。以DON单抗为靶分子,四轮固相淘选噬菌体随机环七肽库,筛选出阳性噬菌体,并通过ELISA实验检测其特异性。对阳性克隆的DNA进行测序,取序列不同的阳性克隆,分别建立阳性噬菌体与DON标准品的竞争抑制曲线。对30个噬菌体单克隆进行活性测定,结果显示能与DON单抗结合的噬菌体克隆有28株,其中的22株能被DON标准品阻断与DON单抗结合。DNA测序结果显示,21株阳性噬菌体的序列是PFPNHPY,另一株的序列是TPWTQHL。其相应噬菌体与DON毒素标准品的竞争曲线结果显示,8号噬菌体建立的竞争抑制曲线线性范围是0.0292⁰.636μg/mL,IC₅₀为0.169μg/mL;4号噬菌体建立的竞争抑制曲线线性范围是0.0124⁰.271μg/mL,IC₅₀为0.058μg/mL。在随机环七肽库中筛选到TPWTQHL和PFPNHPY两个DON模拟表位,可替代DON毒素标准品,建立DON的免疫学无毒检测技术。

Abstract: Screening of mimetic epitopes of deoxynivalenol by using random Ph.D-7 library.DON monoclonal antibody as the target molecule, and Ph.D-7 library had been panned for 4 times. The positive phage was screened out and tested by ELISAspecificity.The activity of 30 phage monoclones was determined by indirect ELISA and indirect competitive ELISA. It showed that 28 phages could combined with DON monoclonal antibody and 22 of them were able to compete with DON standards for binding to DON m Abs.The results of DNA sequencing showed sequence of 21 was PFPNHPY, and the another one was TPWTQHL.The competition curve of corresponding phage and DON toxin standard product showed inhibition curve for the linear range of NO.8 phage was 0.0292⁰.636 μg/mL and IC₅₀ was 0.169 μg/mL, and NO.4 phage was 0.0124⁰.271 μg/mL, IC₅₀ was 0.058 μg/mL.TPWTQHL and PFPNHPY were obtained from Ph.D-7 phage display peptide library.The competitive inhibition curves of phage and DON were established respectively.Preliminary validation shows that the DON toxin standard can be replaced by two DON mimotopes and can be established DON immunological non-toxic detection techniques.