全人源抗体基因库的构建及抗表皮生长因子受体单链抗体的筛选

Construction of a human antibody DNA library and selection of anti-epidermal growth factor receptor single- chain antibodies

目的从肿瘤患者外周血中构建全人源单链可变区抗体基因库，并采用真核核糖体展示技术高效筛选全人源抗表皮生长因子受体（epidermal growth factor receptor，EGFR）单链抗体。方法采用PCR技术从52例晚期肿瘤患者外周血中构建大容量的全人源抗体基因库。针对EGFR抗原靶标，应用真核核糖体展示技术对该基因库进行高效富集筛选。通过大肠埃希菌表达体系对回收的基因库进行克隆、表达，并鉴定抗体活性。结果构建的全人源抗体基因库库容估算为4．3×1013。分子。对该基因库进行3轮富集筛选后，获得EGFR富集的单链抗体基因库。对回收基因库进行克隆、表达，并随机鉴定了49个克隆，获得结合力为10-8～10-7mol／L的2株全人源抗EGFR单链抗体。结论利用肿瘤患者外周血，结合核糖体展示技术可以较快获得具有医用价值的全人源单链抗体。

Objective To construct a fully human single-chain variable fragment （scFv） DNA library and select anti-epidermal growth factor receptor （EGFR） scFvs using ribosome display technology. Methods A fully human scFv library was constructed with peripheral blood samples collected from 52 advanced cancer patients. Eukaryotic ribomme display was used to screen anti-EGFR scFvs from the library. The selected scFvs were then expressed in E. coli and purified. Their binding affinities were validated by ELISA. Results The size of constructed human seFv DNA library was 4.3×10-13 After 3 rounds of selection, 49 unique clones were recovered. The top two binders were further purified and their binding affinities were determined by ELISA to be 10 -8-10-7 mol/L. Conclusion Ribosome display is an efficient screening method to obtain high affinity scFvs that may serve as potential therapeutic candidates.