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立足US28的广谱趋化因子结合活性,寻找拮抗剂多肽的先导物.通过预测US28的结合模拟位设计多肽序列,合成相关多肽,再利用自建的25种趋化因子随机构成的噬菌体12肽库筛选结合模拟位,竞争性ELISA方法验证,从而获得30个阳性克隆,测序并推导氨基酸序列,得到7种序列:GSESLNAHCALW、EIDGFNAHCALL、VIARLNAHCALR、ATECLNAHCALW、VIESLNAHCALW、DNGSINAHCALL及VKKTLNAHCALR.所有序列富含疏水氨基酸,其中8个克隆有LNAHCAL保守序列.采用趋化抑制实验检测多肽对生理性趋化因子引起的细胞迁移活性的影响,流式细胞仪检测细胞内钙离子浓度变化.结果表明,利用人源趋化因子序列构建肽库并筛选有效序列的方法取得成功,筛选出的阳性克隆保守序列LNAHCAL可以模拟HumanMIP-1β与US28N端的结合位,从而与合成多肽H22结合,多肽H22可以阻断受体结合生理性趋化因子形成的趋化作用,本身不引起趋化运动,不影响胞内信号转导和细胞自然活性,具有广谱趋化因子拮抗剂的可能性.
Based on the broad-spectrum chemokine binding activity of US28, the leader of the antagonist polypeptide was searched for.Predicting the polypeptide sequence by predicting the binding mimic of US28 and synthesizing the related peptide, the phage 12 peptide randomly formed by self-built 25 chemokines 30 positive clones were obtained by sequencing, screening and deducing amino acid sequence, and got seven kinds of sequences: GSESLNAHCALW, EIDGFNAHCALL, VIARLNAHCALR, ATECLNAHCALW, VIESLNAHCALW, DNGSINAHCALL and VKKTLNAHCALR.All the sequences were rich in hydrophobic amino acids , Of which 8 were cloned with LNAHCAL conserved sequence.The chemotactic inhibition assay was used to detect the effect of peptides on cell migration induced by physiological chemokines and the change of intracellular calcium concentration was analyzed by flow cytometry.The results showed that using human- LNAHCAL, a conserved sequence of positive clones screened out, can mimic the binding site between HumanMIP-1β and US28N to bind to the synthetic peptide H22. The polypeptide H22 can block the binding of the receptor to the physiological Chemotactic chemotaxis formation, itself does not cause chemotaxis, does not affect Intracellular signal transduction and cellular activity naturally, the possibility of having a broad spectrum chemokine antagonist.