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目的应用噬菌体随机12肽库、DNAStar分析软件、亲和实验及竞争抑制实验初步定位脂多糖结合蛋白(lipopolysaccharide binding protein,LBP)与CD14的结合位点。方法以CD14为筛选分子,联合应用噬菌体肽库展示技术、噬菌体ELISA鉴定、LBP竞争抑制实验及DNA测序推导LBP/CD14结合位点的展示肽序列,采用DNAStar分析软件比对所获展示肽序列与LBP一级结构,初步定位LBP/CD14结合位点并人工合成其模拟肽。采用ELISA检测该模拟肽与CD14的亲和力及与LBP的竞争抑制活性。结果DNAStar比对结果表明,经噬菌体肽库筛选获得的8条展示肽氨基酸序列与LBP第252~263位氨基酸有一定相似性。LBP第252~263位氨基酸具有结合位点的特性,即有良好的亲水性、可及性、可塑性及抗原性,该部位可能是LBP/CD14的结合位点。体外活性实验表明,模拟肽FHRNHRSPVTLL可与CD14有良好的亲和力,有较强的与LBP竞争结合CD14的活性。结论LBP第252~263位氨基酸的模拟肽FHRNHRSPVTLL与CD14有良好的亲和力及有较强的与LBP竞争结合CD14的活性,具有LBP/CD14结合位点的生物学特性,LBP/CD14结合位点初步定位在该区域。
OBJECTIVE: To initially locate the binding site of CD14 with lipopolysaccharide binding protein (LBP) using phage display random peptide library, DNAStar analysis software, affinity experiments and competition inhibition experiments. Methods CD14 was used as a screening molecule, and the phage displayed peptide peptide library, phage ELISA assay, LBP competition inhibition assay and DNA sequencing were used to deduce the displayed peptide sequence of LBP / CD14 binding site. DNAStar analysis software was used to compare the displayed peptide sequence with LBP primary structure, the initial positioning of LBP / CD14 binding sites and artificial synthesis of its simulated peptide. The affinity of the peptidomimetic to CD14 and the competitive inhibition with LBP were detected by ELISA. Results The results of DNAStar alignment showed that the amino acid sequences of the eight displayed peptides obtained by the phage peptide library screening showed a certain similarity with the amino acids 252 to 263 of LBP. The amino acids 252 to 263 of LBP have the characteristics of binding sites, that is, have good hydrophilicity, accessibility, plasticity and antigenicity, which may be the binding site of LBP / CD14. In vitro activity tests showed that the mimic peptide FHRNHRSPVTLL has good affinity with CD14 and has strong competition with LBP for CD14 activity. CONCLUSION: The mimic peptide FHRNHRSPVTLL of amino acids 252 to 263 of LBP has good affinity with CD14 and a strong competitive binding with CD14 on LBP. It possesses the biological characteristics of LBP / CD14 binding site and the initial binding site of LBP / CD14 Locate this area.