中国免疫学杂志2012，Vol.28Issue(2)：137-141,160,6.DOI:10.3969/j.issn.1000-484X.2012.02.009
抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定
Preparation and characterization of recombinant VHH antibody against H5N1 hemagglutinin from avian influenza virus
摘要
Abstract
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 a-vian influenza virus was expressed in E. Coli BL2l( 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.关键词
H5N1禽流感病毒/重链可变区抗体/包涵体复性/活性鉴定Key words
Avian influenza A ( H5N1 ) virus/ Variable domain of heavy chain antibody/ Refolding of proteins from inclusion bodies/ Determination of bioactivity分类
医药卫生引用本文复制引用
夏立亮,吴标,程亚庭,包文静,赵国屏,王颖..抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定[J].中国免疫学杂志,2012,28(2):137-141,160,6.基金项目
本文受国家863 项目(2006AA02A252) 和上海市教委第五期重点学科(J50207) 资助 （2006AA02A252）
