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目的探索利用天然抗角蛋白自身抗体(AKautoAb)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AKautoAb,然后进行生物素标记;用生物素化的AKautoAb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,AKautoAb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AKautoAb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AKautoAb所识别的角蛋白模拟表位。
OBJECTIVE: To explore a method to screen the random peptide library presented by bacteriophage using the natural anti-keratin autoantibody (AKautoAb) to obtain a functional keratin mimotope in vivo. Methods AKautoAb was isolated and purified from normal human mixed serum using keratin affinity chromatography column and then biotinylated. The phage-presented random 15 peptide library was subjected to 3 rounds of panning with ELISA and biotinylated with biotinylated AKautoAb Twenty-three positive clones were picked for DNA sequencing, the data obtained were analyzed and competition experiments performed. Results DNA sequencing analysis showed that AKautoAb specifically recognized 3 epitope regions: SLSPMPTTNRR, PPLIPIPLRTM and TTPRPPMRIMLPRIT, of which SLSPMPTTNRR may contain dominant epitopes; phages carrying these short peptides could specifically bind to AKautoAb and keratin could block this Specific binding reaction. Conclusion The keratin mimotopes recognized by AKautoAb were successfully obtained by using phage random peptide library technology.