抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定

目的:获得具有中和活性、高特异性和稳定性的抗H5N1禽流感病毒血凝素蛋白(HA)的羊驼重链单域(VHH)抗体。方法:利用pET-22b表达载体诱导表达抗H5N1禽流感病毒HA VHH抗体蛋白,以包涵体形式表达的VHH抗体蛋白采用最优复性方法进行复性后,获得高纯度的VHH抗体,分别采用ELISA法鉴定VHH抗体的亲和力和热稳定性,采用血凝抑制实验鉴定抗体的特异性和体外中和活性。结果:经复性的抗H5N1禽流感HA VHH抗体对H5N1禽流感病毒HA具有良好的特异性。通过对三种不同复性方法比较,利用柱上复性的VHH23抗体具有较好的热稳定性,亲和力为9.1×10-7mol/L,同时对H5N1禽流感病毒HA具有良好的体外中和活性。结论:实验结果表明通过原核表达获得具有较好中和活性、特异性及稳定性的抗H5N1禽流感病毒VHH抗体,为进一步开展抗体的体内病毒中和试验奠定良好基础。

Preparation and characterization of recombinant VHH antibody against H5N1 hemagglutinin from avian influenza virus

Objective:To prepare and characterize camelid VHH antibody against hemagglutinin from H5N1 avian influenza virus.Methods:Recombinant expression vector pET-22b-VHH23 was constructed and VHH antibody against hemagglutinin from H5N1 avian influenza virus was expressed in E.coli BL21(DE3) strain by IPTG induction.As VHH antibody protein was expressed in inclusion body,there different refolding methods were compared for the refolding of VHH antibody in which the optimal methods were adopted for preparation of VHH antibody.The activity and thermal stability of VHH antibodies were tested by ELISA while hemagglutination-inhibition assay was perfomed to determine the specificity and neutralizing activity.Results:By using on-column refolding procedure,VHH antibody has been obtained with highest yield and good quality.This antibody specifically recognized hemagglutinin derived from H5N1 avian influenza virus as well as in vitro inhibition against hemagglutinin-mediated red blood cell agglutination.The affinity constant of VHH antibody was 9.1×10-7 mol/L as determined by ELISA.More interestingly,VHH antibody displayed high thermal stability.Conclusion:The results indicated that VHH antibody against hemagglutinin derived from H5N1 avian influenza virus has been obtained with high activity,specificity as well as good thermal stability by using prokecyte expressing system,which provides basis for future functional study in vivo.