重组CPTα的亲和纯化及其单克隆抗体的制备  

Affinity purification of recombinant Clostridium perfringens type A toxin alpha(CPTα)and preparation of its monoclonal antibody(mAb)

作　　者：王景林[1] 杨利敏[2] 吴东林[3] 康琳[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071 [2]内蒙古大学生命科学学院生物系,呼和浩特010021 [3]解放军军需大学军事兽医研究所,长春130062

出　　处：《军事医学科学院院刊》2004年第4期308-310,共3页Bulletin of the Academy of Military Medical Sciences

摘　　要：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 antibody (mAb)against Clostridium perfringens type A toxin alpha (CPTα) and identify its specific biological activity. Methods: 6×his-Tagged CPTα, expressed by genetically engineered strain of E.coli M15/pQE30-CPTa, was purified by one-step method using Ni-NTA affinity chromatography and used for immunizing BALB/c mice. The splenocytes of immunized mice were fused with myeloma cells Sp2/0 and selectively cultured by HAT medium. Hybridoma cells were screened by indirect ELISA and limited dilution was used to clone. Results: One hybridoma cell line 4E2 stably secreting a specific mAb against CPTα was obtained and identified. Identification of subclass showed that mAb 4E2 belonged to IgG1. ELISA showed that mAb 4E2 had a specific affinity for both the rCPTα and native CPTα, and it protected mice from 2 MLD of CPTα challenge. Conclusion: The neutralizing mAb 4E2 obtained revealed a potential diagnostic and therapeutic perspectives for CPTα as well as development of epitope-based vaccine.

关 键 词：A型产气荚膜梭菌 毒素α 亲和纯化 单克隆抗体 

分 类 号：R392[医药卫生—免疫学]