抗炭疽保护性抗原人-鼠嵌合抗体在CHO细胞中的表达和活性研究  

Expression and biological activity analysis of human-mouse chimeric antibody against anthrax protective antigen

作　　者：李冰[1] 李建民[1] 张军[1] 徐俊杰[1] 刘树玲[1] 任军[1] 张金龙[1] 付玲[1] 侯利华[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2009年第12期1069-1074,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家高技术研究发展计划（2006AA02A232）

摘　　要：目的在CHO细胞中表达抗炭疽芽孢杆菌保护性抗原（rPA）人-鼠嵌合抗体，并对其活性进行分析。方法RT—PCR克隆具有中和活性鼠源单克隆抗体的轻、重链可变区基因，分别与人Kappa型轻链恒定区、IgG1重链恒定区基因融合后构建了轻重链嵌合抗体基因。将轻重链嵌合抗体基因克隆表达载体上，构建了嵌合抗体的双启动子表达载体pSecTag-5E1。将pSecTag-5E1转染CHO—dhfr（DG44）细胞，撤掉胸腺嘧啶和次黄嘌呤并不断提高氨甲蝶呤（MTX）的浓度，筛选表达量高的克隆，扩大培养，收集上清，并纯化，用纯化的嵌合抗体进行SDS—PAGE、Westernblot、抗原结合活性、体外细胞保护试验和动物试验。结果在MTX的浓度为5×10^-8mol／L时，获得稳定表达细胞株，表达量为18mg／L。结论成功在CHO—dhfr（DG44）细胞中表达了具有中和活性的抗rPA人-鼠嵌合抗体，为制备抗炭疽的被动免疫制剂奠定了良好的基础。Objective To express human-mouse chimeric antibody against anthrax protective antigen and to analyze its biological activities. Methods A new mammalian bipromoter expression vector was constructed with dihydrofolate reductase（DHFR） gene as the selection and complication marker. First, the light and heavy chain variable region gene of the monoclonal antibody 5E1 were cloned by RT-PCR, at the same time the human IgG1 heavy chain constant region gene and kappa type constant region gene were cloned. Next, the human-mouse chimeric antibody genes were synthesized by fusion PCR. Then, the human-mouse chimeric antibody gene were inserted into MCS of pSeeTag and B1 to construct pSecTag-5E1L and B1-5E1H, respectively. Finally, heavy chain expression cassette excised from the B1-5E1H with Bgl Ⅱ/BamH Ⅰ was further cloned into the Bgl Ⅱ site of the pSeeTag-5EIL to construct pSecTag-5E1. Plasmid pSecTag-5E1 was transfected into CHO （dhfr） engineering cells and high production cell clones that were screened by enhancing MTX concentration. After collecting medium and purifying chimeric antibody with affinity chronmtogram, purified chimeric antibody was analyzed by SDS-PAGE, Western blot. Results A stable and high production cell line was acquired at MTX concentration 5 × 10^-8 mol/L. Conclusion The human-mouse chimeric antibodies were successfully expressed in CHO cells.

关 键 词：炭疽 人-鼠嵌合抗体 CHO细胞 

分 类 号：R686[医药卫生—骨科学]