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目的 :制备抗A型产气荚膜梭菌毒素α(CPTα)单克隆抗体 (mAb)并鉴定其生物学特性。方法 :利用工程菌株E .coliM15 /pQE30 CPTα表达的重组A型CPTα带有 6个组氨酸 (6×his)标签的特性 ,经Ni NTA螯合亲和层析方法纯化后 ,用纯化的重组CPTα免疫BALB/c鼠 ,以常规杂交瘤细胞融合技术、HAT选择性培养和间接ELISA进行筛选 ,并分析其亚类及中和保护作用。结果 :获得一株能稳定分泌抗CPTα的mAb杂交瘤细胞株 4E2 ,其分泌抗体亚类为IgG1。该mAb与重组CPTα和天然CPTα均可发生特异性反应 ,并可保护小鼠抵抗 2倍最小致死剂量CPTα的攻击 ,属中和性抗体。结论 :应用纯化的重组CPTα成功地获得了特异性的中和抗体mAb 4E2 ,为A型CPTα诊断抗体和治疗性抗体的研制奠定了基础。
Objective: To prepare monoclonal antibodies against Clostridium perfringens alpha (CPTα) type A (mAb) and to identify their biological characteristics. Methods: The recombinant A CPTα expressed in engineering strain E.coli M15 / pQE30 CPTα was characterized by 6 histidine (6 × his) tag. After purified by Ni NTA chelate affinity chromatography, purified recombinant BALB / c mice were immunized with CPTα, and selected by conventional hybridoma cell fusion technique, HAT selective culture and indirect ELISA, and analyzed their subclass and neutralization protection. RESULTS: Obtained a mAb hybrid mAb cell strain 4E2 that can stably secrete anti-CPTα, and its secretory antibody subclass was IgG1. The mAb specifically reacts with both recombinant CPTα and native CPTα and protects mice against two-fold lethal dose of CPTα, a neutralizing antibody. Conclusion: The purified recombinant CPTα was successfully used to obtain mAb 4E2, a specific neutralizing antibody, which laid the foundation for the development of diagnostic antibodies and therapeutic antibodies for type A CPTα.