羊驼抗H5N1血凝素重链可变区-人IgGFc段嵌合抗体的制备和鉴定

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。

Preparation and characterization of recombinant Llama VHH-human IgGFc chimeric antibody against H5N1 hemagglutinin from avian influenza virus

To prepare and characterize llama variable domain of heavy chain of heavy-chain antibody-human IgG1Fc(VHH-hFc) chimeric antibody against hemagglutinin from H5N1 avian influenza virus,recombinant expression vector pET-22b-VHH23-hFc was constructed and VHH23-hFc chimeric antibody was expressed in E.coli BL21(DE3) strain by IPTG induction.As VHH23-hFc antibody was accumulated in inclusion bodies,two different refolding methods,dialysis and on-column refolding,were compared for the refolding efficacy and the optimal method was adopted for preparation of VHH23-hFc chimeric antibody.The activity and thermal stability of VHH antibodies were tested by ELISA.By using dialysis refolding procedure,VHH23-hFc chimeric antibody has been obtained with higher yield and good quality.The affinity constant of VHH23-hFc chimeric antibody was 2.24×106 mol/L as determined by ELISA.VHH23-hFc chimeric antibody also displayed good thermal stability.The half-life span of VHH23-hFc chimeric antibody in mice was up to 35 hrs,which is comparable to conventional chimeric antibodies.Taken together,our results indicated that VHH23-hFc chimeric antibody against hemagglutinin derived from H5N1 avian influenza virus has been obtained with high activity,good thermal stability as well as longer half-life span,which provides basis for future functional study both in vitro and in vivo.