摘要
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。
ObjectiveTo investigate the production and characterization of the monoclonal antibody against C5a anaphylatoxin receptor(CD88).MethodsBased on the relationship between the secondary structure and it’s functions of the C5a anaphylatoxin receptor(CD88),the predominant B cell epitope of the C5aR was designed on the computers.Position 9～30 of the C5aR,called as P22 was synthesized using fluorenyl methoxy carbon y1 protected,benzotriazol l Yl oxy tris pyrrolidino phosphonium hezafluorophosphate activated amino acids and an automatic peptide synthesizer.Peptide purity was confirmed by HPLC and identification by caplilliary electrophoresis.The purity of the P22 was 95.19%.McAb generated from hybridoma E3 with specificity for the C5aR(9～30) was obtained by the i.p.immunization of Balb/c mice with 100 μg of P22 conjugate in CFA.The culture supernatants of the hybridoma E3 were secreted by solid phase ELISA techniques employing the C5aR(9～30)peptide,the peptide specific McAb was purified by immunoaffinity chromatography on peptide resin columns.ResultsThe titers of this McAb in culture supernatants and ascites fluid were 1∶32～1∶128,1∶10 -4 ～1∶10 -6 ,respectively.Results from western blotting using reducing agent suggest E3 reacts to a 45 kd bland.The affinity contents of the E3 was 2.5×10 5.ConclusionThe peptide of the B cell epifope of the C5aR has it’s immunoantigence. It could be used to produce monoclonal antibodies.It is also important for studying the interactions between C5a and C5aR,and for ALI,ARDS. [
出处
《免疫学杂志》
CAS
CSCD
北大核心
2000年第4期294-296,303,共4页
Immunological Journal
基金
国家自然科学基金!资助项目 (39770 315 )
