一种可用于赭曲霉毒素免疫检测小分子多肽OTA-1的筛选与鉴定

The Screening and Identification of Small Molecule Peptides OTA1 for Immunoassay of Ochratoxin

目的本研究首次筛选得到一种能够与赭曲霉毒素抗体特异性结合的小分子多肽(OTA-1)序列(QFQLHSMPPTSL),并利用该多肽进行中药材赭曲霉毒素免疫检测研究。方法从噬菌体随机展示12肽库中,经过3轮生物淘选后,随机挑选了30个克隆,其中29个克隆序列与OTA-1完全一致,并通过滴度测定和Western blotting实验,对OTA-1多肽与赭曲霉毒素抗体的亲和力进行了验证。结果通过竞争性ELISA方法,确定了最佳包被浓度和噬菌体浓度,分别为0.5μg/孔和4×1010 pfu/mL。与商品化的ELISA检测试剂盒相比较,本研究开发的噬菌体ELISA检测方法可以用于中药材赭曲霉素的检测。结论表面展示有OTA-1多肽的噬菌体与赭曲霉毒素是一种竞争关系,都能够识别赭曲霉毒素抗体并与其结合,这为真菌毒素的免疫检测提供了一种新的策略。

Objective This study is the first time to screen the small molecule polypeptide(OTA-1)sequence(QFQLHSMPPTSL)that can specifically bind to ochratotoxin antibody,and to study the immunoassay of ochratotoxin.Methods After three rounds of bio-screening,30 clones were randomly selected,of which 29 clones were completely identical with OTA-1,and the affinity and specificity of the small molecule peptide OTA-1 were verified by titer determination and Western blotting experiments.Results After a competitive ELISA analysis,the optimal inclusion concentration and phage concentration were 5.0μg/well and 4×1010 pfu/mL.Competed with industrial ELISA kit,the method based on phage ELISA could used to detect ochratotoxin in Chinese medicinal herbs.Conclusion So both the phage displayed with OTA-1 peptides and ochratotoxin can bind with anti-ochratotoxin antibodies,which showed a competitive relationship between them.This provide a new strategy for ochratoxin detection in the future.