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目的从蛋 白质结构与功能的关系出发,探讨C5aR与其配体C5a的结合位。方法按分子设计理论,采用亲水性方案寻找C5aR胞外区高亲水性区域,Fmoc 方案人工合成 C5aR第9～ 30位氨基酸基序(P22肽),经高压液相色谱纯化,毛细管电泳鉴定。结果 合成多肽(P22)的纯度为95.19%,每次缩合的平均效 率为99.78%;能与anti-C5aR McAb(S5/1,Serotic 公司)有效地结合,酶联O D490值极显著高于同浓度对照多肽C20;还可被配体rh-C5a (10 μg/L )明显抑 制而降低其酶联OD值(P<0.05)。此外,10 μg/L P22还可抑制rh-C5a 所 致U937细胞胞浆Ca~`(2+)升高(P<0.01)。结论从C5aR分子中寻找C5a结合位基序并予以人工合成,可中和C5a的过敏毒素作用, 为治疗C5a及其相关疾病进行新型的药物设计。
Objective To investigate the relationship between C5aR and its ligand C5a in terms of the relationship between protein structure and function. Methods According to the molecular design theory, the hydrophilic region was used to find the extracellular region of C5aR with high hydrophilicity. The amino acid motif (P22 peptide) of C5aR was synthesized by Fmoc protocol and purified by high pressure liquid chromatography (HPLC) . Results The purity of synthetic peptide (P22) was 95.19% with an average efficiency of 99.78% for each condensation. The binding efficiency of anti-C5aR McAb (S5 / 1, Serotic) was significantly higher than that of Concentration of control peptide C20 was also significantly inhibited by ligand rh-C5a (10 μg / L) and decreased its OD (P <0.05). In addition, 10μg / L P22 also inhibited rh-C5a-induced cytoplasmic Ca ~ (2+) increase in U937 cells (P <0.01). Conclusion The search for C5a binding motif from C5aR molecule and its synthetic synthesis can neutralize the anaphylatoxins of C5a and make new drug design for the treatment of C5a and its related diseases.