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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。
OBJECTIVE: To humanize the murine monoclonal antibody (mAb) framework region by using the antibody “framework region remodeling” technology to prepare humanized scFv and test its activity. Methods: After obtaining the variable region gene of anti-Bacillus anthracis protective antigen mouse mAb (5E1), keeping the complementarity-determining region (CDR) of mouse mAb unchanged, the human sequence homologous to the framework region was selected to replace the mouse The framework region of the mAb retains individual key murine residues. Humanized scFv was constructed by fusion PCR technique and expressed in E. coli. The expressed product existed in the form of inclusion body, and denatured, nickel-affinity chromatography and renaturation to obtain refolded soluble protein. The purified products were tested by SDS-PAGE, ELISA and anthrax toxin neutralization activity. Results: The humanized sequence has antigen-binding activity consistent with murine scFv and cell-neutralizing anthrax toxin activity. Conclusion: The humanized antibody gene sequence with function was obtained, which laid the foundation for the expression of full-molecule humanized antibody with neutralizing anthrax toxin activity.