利用噬菌体肽库技术获得与人Fas结合的多肽基序

Use a phage display library to identify oligopeptides binding to the Fas

目的 利用噬菌体随机肽库技术获得与人Fas胞外区结合的多肽及其多肽基序 ,观察多肽基序的生物学功能。方法 以人Fas胞外区与IgGFc段的融合蛋白Fas .Fc为筛选配基 ,筛选噬菌体随机九肽库 ;微量淘洗与ELISA相结合鉴定阳性克隆 ,DNA测序和分析。化学合成多肽进行竞争性ELISA以及细胞增殖抑制实验。结果 经过 4轮亲和筛选 ,微量淘洗鉴定 ,获得 4 2个阳性克隆 ;固定ELISA实验显示筛选到的噬菌体短肽能与Fas .Fc特异性结合 ,并呈剂量依赖关系 ;随机选取 13个阳性克隆进行DNA测序 ,其序列及出现几率分别为 :PRKARVDTS(2 / 13)、YKKKSLQVQ (2 / 13)、YKKKSMLQA(2 / 13)、SRKKYDQYA(4/ 13)、YARKIKPTA(2 / 13)和ARKKTEGAG(1/ 13)。经多重序列分析 ,获得多肽基序 : R/KKK A。在ELISA和竞争性ELISA实验中 ,化学合成多肽EGEFYKKKSM LQADPAK (P3)可抑制Fas与抗人Fas单抗Apo 1的结合 ,且呈剂量效应关系 ;P3不能抑制Fas与FasL的结合。细胞增殖实验表明 ,多肽可抑制Jurkat细胞增殖 ,且随多肽剂量的增加而加强。多肽与单抗Apo 1联合作用对Jurkat细胞增殖的影响与单独使用P3没有明显差别。结论 通过噬菌体随机肽库技术获得与Fas结合的多肽及其多肽基序 ,它们可能模拟了抗Fas抗体Apo 1对Fas的结合位点 。

Objective To obtain the oligopeptides binding to the Fas.Fc from a phage display random peptide library and to explore their bio logical function. Methods Phage display random nonapeptid e library was immunoscreened with purified Fas.Fc. After 4 rounds of biopanning, positive isolated clones were detected by micropanning and ELISA. 13 clones ran dom selected from positive clones were sequenced, one of which was synthesized a nd analyzed by competitive ELISA and cell proliferation assays. Resul ts Forty-two positive clones specifically bound with Fas.Fc in a do se-dependent manner. Thirteen clones of them were sequenced and the foreign pep tides and frequencies were as follows: PRKARVDTS(2/13), YKKKSLQVQ(2/13), YKK KSMLQA(2/13), SRKKYDQYA(4/13), YARKIKPTA(2/13) and ARKKTEGAG(1/13). All of them contained one motif: *R/KKK****A. One of them was synthesized and purif ied. The result of competitive ELISA showed that synthetic pepti de P3 inhibited the binding of Fas with Apo-1 dose-dependently but had no inhi bition to the interaction of FasL and Fas. The peptide P3 inhibited the Jurkat c ell proliferation in dose-dependent manner as demonstrated by cell proliferatio n assays. However, there is no difference between the inhibition of Jurkat cells proliferation by peptide P3 with Apo-1 McAb and one by peptide P3 only. Conclusion Six peptides specifically binding with Fas.Fc were successfully obtained from a phage display random peptide library containing one peptide motif, which simulated the binding site of anti-Fas antibody Apo-1 to Fas. The results provide useful experimental and structure data for the design of peptide drugs to treat Fas-associated diseases. [