噬菌体展示全人源抗GPC3的单链抗体的筛选及鉴定

发布时间：2019-05-20 00:57

【摘要】：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。
[Abstract]:The single chain antibody targeting phosphatidylinositol 3 (GPC3) was obtained by screening the whole human bacteriophage single chain antibody library, and the biological activity and antigenic epitope of the single chain antibody were identified. After four rounds of "binding-eluting-amplification", the targeting and integrity of antibody sequences were analyzed by bacteriophage ELISA and IMGT database. The gene sequence of single chain antibody was digested and ligated with the expression vector p ET-22b, and the recombinant vector was transformed into E. coli E.coli Rosetta (DE3). The antibody was expressed and purified by Ni~ (2) column affinity chromatography. The molecular weight of single chain antibody was identified by SDS-PAGE and Western blot. The affinity constant of antibody to antigen was obtained by ELISA and SPR assay. The binding ability of antibody to cell membrane receptor was analyzed by flow cytometry. Four single chain antibodies (1F7, 1D7, 1D4 and 1B10) were obtained with good targeting and affinity, among which 1F7 single chain antibodies had the highest affinity and binding ability. In this study, anti-GPC3 single chain antibody laid a foundation for the development of new immunotherapeutic drugs such as bicharacteristic antibodies and immunotoxins.
【作者单位】：中国药科大学生命科学与技术学院;
【基金】：国家自然科学基金面上项目资助(81473125) 江苏省自然科学基金资助项目(BK20161459) 西比曼生物科技(上海)有限公司项目资助
【分类号】：R392-33

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