天然抗角蛋白自身抗体识别的角蛋白模拟表位的研究

目的 探索利用天然抗角蛋白自身抗体(AK auto Ab)淘筛噬菌体递呈的随机肽库,获得在体内具有功能活性的角蛋白模拟表位方法。方法 用角蛋白亲和层析柱从正常人混合血清中分离并纯化AK auto Ab,然后进行生物素标记;用生物素化的AK auto Ab对噬菌体递呈的随机15肽库进行3轮淘洗并进行ELISA检测;挑取23个阳性克隆进行DNA测序,分析所获数据并进行竞争实验。结果 DNA测序分析表明,AK auto Ab特异识别3个抗原表位区:SLSPMPTTNRR、PPLIPIPLRTM和TTPRPPMRIMLPRIT,其中SLSPMPTTNRR可能含有优势表位;携有这些短肽的噬菌体可与AK auto Ab特异结合,角蛋白可阻断这种特异的结合反应。结论 利用噬菌体随机肽库技术成功地获得了AK auto Ab所识别的角蛋白模拟表位。

Study on the Epitope Mapping Which Binds to Polyclonal Anti- keratin Autoantibodies Using a Phage Random Peptide Library

Objective To design and testify a novel strategy for acquiring mimetic epitope mapping by screening for a phage random peptide library using polyclonal anti keratin autoantibodies (AK auto Ab). Methods AK auto Ab were isolated and purified from pooled human sera by keratin affinity column in which keratin had been linked with CNBr Sepharose 4B,then biotinylated by the biotin ester. A 15 mer phage random peptide library was biopanned for 3 cycles and positive clones were identified by ELISA,competition assay and DNA sequencing. ResultsBy sequence comparison 23 positive clones were selected randomly and three epitopes were confirmed. Among the three epitopes SLSPMPTTNRR was the dominant epitope. The phages carrying positive clones reacted with AK auto Ab specifically and keratin could prevent interaction between AK auto Ab and positive phages. Conclusion The designed strategy is successfully applied in acquiring epitopes of polyclonal autoantibodies to keratin, which could provide a new approach for the discovery of epitope mapping which binds to natural autoantibodies.