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本研究旨在制备羊驼抗H5N1禽流感病毒的重链抗体可变区-人Fc段嵌合体抗体制备,对所得嵌合抗体进行制备和功能鉴定,为临床应用奠定基础。用pET-22b表达载体构建抗H5N1禽流感病毒羊驼重链可变区(VHH)-人IgG1Fc嵌合基因,以包涵体形式表达VHH23-hFc嵌合抗体蛋白,采用优化的方法复性后,获得高纯度VHH23-hFc嵌合抗体,用ELISA法鉴定嵌合抗体亲和力、热稳定性和小鼠体内的半衰期。结果显示,透析复性后原核表达的抗H5N1禽流感病毒VHH23-hFc嵌合抗体亲和力为2.24×106 mol/L,具有较好免疫学活性,热稳定性也较好,小鼠体内半衰期达到35h,为下一步开展该抗体的体内外病毒中和试验奠定良好基础。
The purpose of this study was to prepare the antibody of variable region-human Fc fragment chimeric antibody of alpaca anti-H5N1 avian influenza virus and to prepare and characterize the chimeric antibody obtained therefrom, which laid the foundation for clinical application. The VHH-human IgG1 Fc chimeric gene of anti-H5N1 strain of avian influenza virus (H5N1) was constructed by using pET-22b expression vector and the VHH23-hFc chimeric antibody protein was expressed as a inclusion body. After renaturation by optimized method, High purity VHH23-hFc chimeric antibody was obtained. The chimeric antibody affinity, thermal stability and half-life in mice were identified by ELISA. The results showed that the affinity of VHH23-hFc chimeric antibody against H5N1 strain was 2.24 × 106 mol / L, which showed good immunological activity and good thermal stability. The in vivo half-life of mice reached 35h , Which lays a good foundation for the virus neutralization test in vitro and in vivo for the next step.