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目的获得有生物功能的小鼠抗人 C5 a R短肽单克隆抗体。方法对 C5 a受体 (CD88)二级结构和 B细胞表位进行分析研究 ,采用 Fmoc方案固相合成 C5 a R N-端第 9～ 30位氨基酸残基的 2 2 -肽 ,以此为抗原免疫 Balb/ c小鼠。结果建立了 1株小鼠抗人 C5 a R短肽杂交瘤细胞系 E3,其平均染色体数目为 10 2条 ,所分泌抗体为 Ig G1,κ型 ,腹水抗体效价为 1×10 - 4 ～ 1× 10 - 6 ,可识别 U 937、人脐静脉内皮细胞 (VEC)和 PMN等表达 C5 a R细胞。 E3单抗的亲和常数 Ka=2 .5× 10 5 ,其结合表位为 C5 a R第 15～ 2 1位氨基酸基序 D1 5 DKDTL2 0 D。结论 B细胞表位多肽具有免疫原性 ,可制作单克隆抗体 ,为 C5 a-C5 a R相互作用的研究以及 AL I、ARDS等 C5 a相关疾病的研究提供实验材料。
Objective To obtain mouse monoclonal anti-human C5 a R peptide with biological function. Methods The secondary structures of C5a receptor (CD88) and B cell epitopes were studied. The Fmoc scheme was used to synthesize the 2 2 - peptide with amino acid residues 9-30 of N-terminal of C5 a R. Antigen immunized Balb / c mice. Results A mouse anti - human C5 a R short peptide hybridoma cell line, E3, was established. The average number of chromosomes was 10 2 and the secreted antibodies were Ig G1 and κ. The antibody titer of ascites was 1 × 10 -4 ~ 1 × 10 - 6, recognizing U 937, human umbilical vein endothelial cells (VEC) and PMN expressing C5 a R cells. E3 monoclonal antibody affinity constant Ka = 2.5 × 10 5, the binding epitope C5 a R 15 ~ 2 1 amino acid sequence D1 5 DKDTL20D. CONCLUSION: B-cell epitope peptides are immunogenic and can be used as monoclonal antibodies for the study of C5a-C5aR interaction and C5a-related diseases such as ALI and ARDS.