利用噬菌体展示技术制备人血管生成素2单链抗体

摘要

Objective To identify and purifyt single-chain antibody against angiopoietin 2 (Ang2-scFv) by phage display technology from non-immune mouse antibody phage display library by purified human angiopoietin2 protein,which will lay foundation for further functional studies of Ang2-scFv.Methods By using purified human angiopoietin2 protein as antigen,Ang2-scFv was screened from the non-immune mice phage display antibody library.The target protein expression was detected by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blotting,and DNA of the 750 bp Ang2-scFv gene was sequenced.Results After three rounds of planning,one positive clone with specifically binding ability to angiopoietin2 protein was obtained.DNA sequencing showed that it contained a complete single-chain antibody fragment.Identification and purification of the Ang2-scFv was realized by SDSPAGE technique.Conclusion We successfully separated and purified the Ang2-scFv protein,which lay the foundation for the further functional studies of the single-chain antibody.%目的 用纯化的人血管生成素2(Ang2)蛋白,通过噬菌体展示技术从非免疫小鼠噬菌体展示抗体库中淘选、鉴定Ang2单链抗体.方法 以纯化的人Ang2蛋白为抗原,对非免疫小鼠噬菌体展示抗体库进行富集和筛选,获得Ang2单链抗体,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot法检测目的蛋白的表达并测序.结果 经过3轮富集和筛选,获得了1个能特异性识别Ang2蛋白的阳性噬菌体克隆,DNA测序表明其含有完整的单链抗体基因片段,大小约750 bp,表达蛋白相对分子质量约33.7×103；通过SDS-PAGE进一步实现Ang2单链抗体(phage-Ang2-scFv)的鉴定.结论 成功分离并鉴定人Ang2-scFv,为进一步的功能学研究奠定了基础.