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

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.