人源特异性抗狂犬病毒二价Fab094-DDD抗体的制备及鉴定

Generation and characterization of a human neutralizing bivalent antibody Fab094-DDD against rabies virus

目的 :利用蛋白激酶A(protein kinase A,PKA)调节亚基(R亚基)的二聚体化功能结构域(DDD)的二聚化功能,制备具有中和活性的人源特异性抗狂犬病毒二价抗体。方法:优化合成linker-C-DDD序列,设计引物扩增抗狂犬病毒抗体Fab094的Fd段和轻链可变区序列(Vκ),通过overlap PCR将Fab094的Fd段和linker-C-DDD基因重组为Fd-DDD,将其和Vκ分别克隆到真核表达载体中,共转染293Free style细胞,表达纯化该抗体。应用SDS-PAGE、Western blot、ELISA、免疫共沉淀、亲和力测定及免疫荧光试验检测该抗体的免疫学特性,用荧光抗体病毒中和试验检测其中和活性。结果:成功构建了全人源抗狂犬病毒二价抗体Fab094-DDD的真核表达载体,获得的二价Fab094-DDD抗体与抗原保持着较好的亲和力,能特异性结合狂犬病毒,中和效价为213.2 U/mg。结论:成功制备了抗狂犬病毒二价Fab094-DDD抗体,具有较高的中和活性,为进一步研发狂犬病治疗性抗体药物奠定了基础,对于其他疾病双表位人源特异性抗体的制备也具有借鉴作用。

Objective：We sought to produce a human specific anti-rabies-virus bivalent antibody with neutralizing activity by using the dimer effect of RII’s dimerization and docking domain（DDD）in protein kinase A（PKA）. Methods：Linker-C-DDD sequence was optimized and synthesized. Fd section and light chain variable region （Vκ） of anti-rabies virus Fab094 were amplified. Fd section of Fab094 and linker-C-DDD gene were recombined using overlap PCR. Fd-DDD and Vκ were cloned into eukaryotic expression vectors and both vectors were transfected into 293 free style cells. Fab094-DDD antibody was expressed and purified. The immunological features of Fab094-DDD was detected by SDS-PAGE,Western blot assay,enzyme-linked immunosorbent assay（ELISA）,co-IP,affinity test and indirect immunofluorescence test. The neutralizing activity of Fab094-DDD was detected by fluorescent antibody virus neutralization test. Results：The results demonstrated that the eukaryotic expression vectors of the human specific anti-rabies virus bivalent Fab094-DDD antibody were successfully constructed. The antibody effectively recognized the antigen and specifically combined the rabies virus,and the neutralization titer of the antibody was 213.2 U/mg. Conclusion：We successfully generated a bivalent Fab094-DDD antibody against rabies virus,and the antibody showed high neutralizing activity. Fab094-DDD antibody could establish the foundation for the treatment of rabies and could be applied to the construction of other bispecific human specific antibodies.