人源化抗炭疽芽胞杆菌保护性抗原单链抗体的设计和表达  

Expression and neutralizing activity analysis of humanized single-chain antibody against anthrax protective antigen

作　　者：李冰[1] 张军[1] 徐俊杰[1] 刘树玲[1] 付玲[1] 李建民[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：《细胞与分子免疫学杂志》2010年第2期145-148,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高科技研究发展计划(863)资助项目(2006AA02A232)

摘　　要：目的:应用抗体"框架区重塑"技术,对鼠单克隆抗体(mAb)框架区进行人源化,制备人源化单链抗体(scFv)并检测其活性。方法:在获得抗炭疽芽胞杆菌保护性抗原鼠mAb(5E1)可变区基因的基础上,保持鼠mAb互补决定区(CDR)不变,选择与框架区同源性最高的人源序列替换鼠mAb的框架区,保留个别关键的鼠源残基。通过融合PCR技术构建人源化的scFv,并在大肠杆菌中进行了表达。表达产物以包涵体形式存在,通过变性、镍柱亲和层析和复性,获得复性后的可溶蛋白。对纯化产物进行了SDS-PAGE、ELISA和抑制炭疽毒素中和活性的检测。结果:人源化后的序列具有与鼠源scFv一致的抗原结合活性和细胞水平的中和炭疽毒素活性。结论:获得了具有功能的人源化的抗体基因序列,为表达具有中和炭疽毒素活性的全分子人源化抗体奠定了基础。AIM：In order to reduce the human anti-mouse antibody（HAMA） response,anti-anthrax protective antigen scFv-5E1 has been humanized.METHODS：The authors have constructed a ＂humanized＂ antibody fragments by grafting the complementarity-determining regions（CDRs） of the murine anti-PA scFv-5E1 to human framework regions which showed maximal homology to the scFv-5E1 sequence.The humanized VH-5E1 and VL-5E1 DNA fragments were then joined by fusion PCR.The expression vectors named pET-hscFv-5E1 was constructed by cloning the humanized 5E1 scFv gene into the Nde I/EcoR I site.The transformed E.coli BL21（DE3） cells were propagated and induced by IPTG.RESULTS：Expression product was found as inclusion body with expected size by SDS-PAGE analysis.Soulable scFv showed binding ability to the protective antigen by ELISA analysis.The purified scFv showed good neutralizing activity in a cell model.CONCLUSION：The humanized scFv exhibited good binding and neutralizing activity.It lays a foundation for the development of therapeutic whole molecular antibody.

关 键 词：单链抗体 免疫原性 人源化 炭疽 

分 类 号：R392.11[医药卫生—免疫学]