人源性抗HBsAg单链抗体在大肠杆菌中的表达及活性测定  

Expression and bioactivity of a human single-chain Fv antibody against the HBsAg

作　　者：任向荣[1] 熊盛[2] 唐永红[1] 粟宽源[1] 陈文吟[1] 郑业华[1] 余宙耀[1] 

机构地区：[1]解放军第458医院全军传染病中心,广州510602 [2]华南理工大学生物工程系,广州510640

出　　处：《中国免疫学杂志》2002年第12期824-827,共4页Chinese Journal of Immunology

基　　金：广州市科委重点攻关项目 ( 97- z -6 5- 0 5 )

摘　　要：目的 :制备有活性的人源性抗HBsAg单链抗体。方法 :应用噬菌体表面呈现技术获得人抗乙型肝炎表面抗原(HBsAg)抗体的Fab片段 ,并将VH 和VL 基因以 (Gly4Ser) 3linker连接成单链 ,插入表达载体pQE40 ,筛选大肠杆菌高表达菌株。结果 :工程菌表达优化试验表明 ,在OD6 0 0 为 0 6时开始诱导 ,持续 6h ,目的蛋白表达量最高可达菌体总蛋白的 31%。包涵体变性后上样镍离子螯合层析柱一步纯化 ,收集目的峰 ,透析复性 ,得纯度为 97%的重组单链抗体 ,其亲和常数为 0 2 3× 10 8mol L。结论 :抗HBsAg单链抗体在大肠杆菌内获得高效表达 ,经变性和复性 ,得到有活性的单链抗体 ,氨基酸组成正确。为进行临床前研究打下基础。Objective:To produce human anti-HBsAg scFv which has activity of HBsAg binding.Methods:The V H and V L gene,amplified from the vector including human Fab fragment against HBsAg which was acquired by phage display,were linked together by(Gly 4Ser) 3 as a single-chain Fv antibody,which was then inserted into the vector pQE-40 and expressed as inclusion body in E.coli M15.Results:The optimal condition of induction was found to induce the culture of recombinant at 0.6 OD 600 for 6 hours.Under this experiment condition,the target protein amounted 31% of the total protein,reaching its highest production.The lysed inclusion body was purified by Ni 2+ charged column and the target protein was refolded by dialysis,so obtained the r-scFv to a purity of 97% with the bioactivity of HBsAg binding.The affinity constant of the refolded scFv was confirmed by non-competed ELISA to be 0.23×108 mol/L.Conclusion:The stable and efficient expression of scFv in M15-pQE40 can produce scFv antibody against HBsAg and this antibody could be the foundation for further clinic research.

关 键 词：大肠杆菌 噬菌体表面呈现 乙型肝炎表面抗原 单链抗体 表达 复性 生物活性 

分 类 号：Q786[生物学—分子生物学]