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使用全人源噬菌体单链抗体文库进行筛选,获得特异性靶向磷脂酰肌醇聚糖-3(GPC3)的单链抗体,并对单链抗体的生物学活性和抗原表位进行鉴定。经过4轮“结合-淘洗-洗脱-扩增”后,利用噬菌体ELISA和IMGT数据库分析抗体序列的靶向性和完整性。单链抗体的基因序列与表达载体p ET-22b进行双酶切并连接,重组载体转化大肠杆菌E.coli Rosetta(DE3),抗体表达后经Ni~(2+)柱亲和层析纯化,通过SDS-PAGE和Western blot对单链抗体分子质量进行鉴定。抗体对抗原的亲和力常数通过ELISA和SPR实验获得,流式细胞术分析其与细胞膜受体的结合能力。经筛选获得4个单链抗体(1F7、1D7、1D4和1B10)具有良好的靶向性和亲和力,其中1F7单链抗体的亲和力和结合能力最高。本课题抗GPC3单链抗体为双特性抗体和免疫毒素等新一类免疫治疗药物的开发奠定基础。
The whole-phage single-chain antibody library was screened to obtain a single chain antibody that specifically targets glypican-3 (GPC3), and the biological activity of the single-chain antibody and the epitope were identified. After 4 rounds of “binding-elute-elute-expand” the phage ELISA and IMGT databases were used to analyze the targeting and integrity of the antibody sequences. The gene sequence of the single chain antibody was double-digested and ligated with the expression vector p ET-22b, and the recombinant vector was transformed into E. coli Rosetta (DE3). The antibody was purified and purified by Ni 2+ column affinity chromatography. The molecular weight of the single-chain antibody was identified by SDS-PAGE and Western blot. Affinity constants of the antibodies against the antigen were obtained by ELISA and SPR experiments, and their binding ability to the cell membrane receptor was analyzed by flow cytometry. Four scFv antibodies (1F7, 1D7, 1D4 and 1B10) were screened for their good targeting and affinity. Among them, 1F7 scFv had the highest affinity and binding capacity. The subject anti-GPC3 single-chain antibody for the development of a new class of immunotherapy drugs such as dual-antibody and immunotoxin lay the foundation.