噬菌体随机肽库淘选桔霉素模拟表位的研究

Selecting Mimotope of Citrinin from Phage Random Peptide Library

目的:从噬菌体随机肽库中淘选模拟桔霉素表位的噬菌体粒子。方法:以抗桔霉素的单克隆抗体为配基,分别免疫亲和淘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7肽库和12肽库,以ELISA方法鉴定阳性克隆,同时进行DNA测序以分析插入的7肽和12肽的氨基酸序列。结果:经过3轮淘选,在7肽库中淘选到20株能与该抗体特异性结合的阳性克隆,在12肽库中淘选到33株能与该抗体特异性结合的阳性克隆,且该结合均能被桔霉素阻断,模拟表位的共有序列为X-组氨酸-赖氨酸-X-X-X-X,X为任意氨基酸。以7肽库中亲和力最强的克隆(P10)建立了竞争ELISA检测方法,线性范围为10～325ng/ml,检测下限为10ng/ml;以12肽库中亲和力最强的克隆(P1)建立了竞争ELISA检测方法,线性范围为10～439ng/ml,检测下限为10ng/ml。结论:噬菌体展示技术可成功淘选到桔霉素模拟表位,高度保守的His和Lys的存在,提示His和Lys在CIT与其配体的结合中可能起重要作用。

Objective： To screen phage particles capable of mimicking citrinin. Methods： An anticiarinin monoclonal antibody was used as ligand. Biopanning was done to screen the mimotope from a phage random 7 peptide library and a phage random 12 peptide library. These libraries were displayed as a fusion protein with the coat protein M of filamentous phage M13. The positive clones were identified by ELISA, and the inserted amino sequences were deduced by DNA sequencing. Results After three rounds of panning, 20 positive clones could bind to the antibody,and the binding could be blocked by free citrinin in the phage random 7 peptide library; After three rounds of panning, 33 positive clones could bind to the antibody.and the binding could be blocked by free citrinin in the phage random 12 peptide library. The common amino sequence of the mimicking epitope was XHKXXXX. A competitive ELISA immunoassay was established with clone P10 of the phage random 7 peptide library, the linear range of the inhibition curves was between 10ng/ml and 325ng/ml; the detecting limitation was 10ng/ml. A competitive ELISA immunoassay was established with clone P1 of the phage random 12 peptide library,the linear range of the inhibition corves was between 10ng/ml and 439ng/ml; the detecting limitation was 10ng/ml. Conclusion： The phage display technique could be successfully applied to screen the mimotope of citrinin. The conserved His and Lys indicate that it maybe play a key role in citrinin receptor binding to citrinin.