【摘要】 目的应用噬菌体随机七肽库筛选与人CD137特异结合的肽序列。方法以重组人CD137作为筛选分子,对噬菌体展示随机七肽库进行亲和淘选。经过5轮淘选后,共随机挑选23个噬菌斑并对其进行了扩增和测序,据此推导随机多肽的氨基酸序列。经夹心ELISA进一步鉴定噬菌体克隆与CD137的结合力。3H-TdR法检测了噬菌体展示多肽对抗CD137Ab刺激的人外周血淋巴细胞增殖反应影响。结果5轮淘选所得的噬菌体回收率逐步提高,显示淘选过程对随机肽库有一定的富集效果。通过对阳性克隆的测序和序列比较分析,得到RPTQGAF、RPTQVAL、RPTQVAF的相似序列和与CD137L具有同源SRS序列的SRSRVRY、HRRPSRS肽序列,ELISA结果显示这5个克隆都有良好的与CD137的结合力。RPTQGAF、RPTQVAL、RPTQVAF和SRSRVRY4个噬菌体展示肽均能在体外抑制抗CD137Ab刺激的淋巴细胞增殖反应。结论通过对噬菌体随机肽库的淘选,得到与CD137结合的相关多肽,为进一步研究CD137与配体结合的特异性位点及其拮抗性多肽药物设计提供了实验依据和结构基础。更多还原

【Abstract】 Objective To screen polypeptides binding to human CD137 by biopanning phage display random heptapeptide library.Methods Phage display random heptapeptide library was screened with recombinant human CD137. After five rounds biopanning, thirty-three positive clones were selected for automated sequencing and the amino acid sequence of polypeptide displayed on phage was deduced. Their affinity to CD137 was quantified by ELISA. ~3H-TdR assay was used to detect the effect of phage display peptides to CD137Ab induced T cell proliferation.Results The enrichment was shown after five rounds of biopanning. Thirty-three positive clones specifically bind to CD137 were screened from the library and sequenced. Similar peptide sequence was found in 4 positive clones. Two peptide sequence SRSRVRY, HRRPSRS have homologous sequence SRSXXRX compared with CD137L. ELISA test showed they all have affinity to CD137. We also gained another five clones that show high affinity binding to CD137 by ELISA test. ~3H-TdR test showed that phage display peptides RPTQGAF, [CM(43]RPTQVAL, RPTQVAF and SRSRVRY suppressed CD137Ab induced T cell proliferation in vitro.Conclusion[CM)]Peptides binding to CD137 were successfully obtained from a phage display random heptapeptide library. The results provide useful experimental and structure data for identification of the binding domain for CD137 and its ligand, and also for the design of peptide drugs to block CD137 costimulation pathway.更多还原