人源抗EB病毒潜伏膜蛋白1特异性基因工程抗体Fab的筛选与特性

Selection and characterization of anti-EBV-LMP1 humanized genetic engineering antibody Fab

目的:应用噬菌体展示技术制备抗EB病毒编码的潜伏膜蛋白1(latert membrare protern 1,LMP1)特异性、高亲和力的全人抗体Fab片段。方法:利用稳定转染表达LMP1的SUNE-1细胞及原核表达的LMP1胞外区蛋白作为抗原对大容量人源Fab抗体库进行差减筛选及固相筛选,经过5轮细胞筛选和3轮固相筛选,随机挑选30个克隆经酶联免疫吸附法鉴定,阳性克隆进行可溶性表达,用LMP1阳性表达的人鼻咽癌细胞株HNE2/LMP1鉴定抗LMP1抗体Fab的结合活性。结果:Western blot、免疫荧光结果显示,从大容量人源Fab抗体库中筛选出1株抗LMP1抗体Fab,细胞ELISA、荧光激活细胞分类术、免疫荧光分析、免疫组化检测结果显示Fab与人鼻咽癌细胞表面LMP1分子有较好的结合活性。结论:从人源Fab抗体库中筛选的Fab抗体能够与鼻咽癌细胞表面LMP1分子特异性结合,为研制用于EBV相关肿瘤如鼻咽癌生物治疗的靶向药物,提供了候选分子。

Objective：To screen anti-EBV-LMP1 Fab of high specificity and affinity from a phage antibody library and identify its binding activity.Methods：Subtractive screening method by alternating SUNE-1 / LMP1 cells and solid-phase from a large capacity Fab phagelibrary.After eight rounds screening,30 positive clones were identified by ELISA test.The positive clones were selected by Western blot for Fab soluble expression in TOP 10F’ and the binding activities of Fab with LMP were analyzed in HNE2 / LMP1 cell lines.Results：A Fab fragment with fine activity to LMP1 was selected,purified and expressed.The anti-LMP1 Fab binding specificity was confirmed by cell ELISA,FACS,immunofluorescence and immunohistochemistry.Conclusion：The anti-LMP1 Fab binding to nasopharyngeal carcinoma（NPC） cells provides a promising candidate for the biotherapy of EBV-related carcinoma.