天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (8): 851-855.doi: 10.11958/20170531

• 细胞与分子生物学 • 上一篇    下一篇

人源性 TRAb Fab 段单克隆抗体的筛选和鉴定

杜晓明 1， 李宁 2， 方佩华 2△   

  1. 1 天津市第四中心医院内分泌科 （邮编 300140）； 2 天津医科大学总医院核医学科
  • 收稿日期:2017-05-02 修回日期:2017-06-25 出版日期:2017-08-15 发布日期:2017-08-15
  • 通讯作者: △通讯作者 E-mail: raysinomail@163.com E-mail:duxiaoming99@aliyun.com
  • 作者简介:杜晓明 （1971）， 女， 博士， 副主任医师， 主要从事甲状腺疾病研究
  • 基金资助:
    天津市卫生局科技重点项目 （2013KR04）

Selection and identification of human monoantibody TRAb Fab fragment

DU Xiao-ming1, LI Ning2, FANG Pei-hua2△   

  1. 1 Department of Endocrinology, Tianjin 4th Center Hospital, Tianjin 300140, China; 2 Department of Nuclear Medicine, Tianjin Medical University General Hospital
  • Received:2017-05-02 Revised:2017-06-25 Published:2017-08-15 Online:2017-08-15
  • Contact: △Corresponding Author E-mail: raysinomail@163.com E-mail:duxiaoming99@aliyun.com

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摘要： 目的 通过噬菌体表面展示技术， 运用已构建的人源性噬菌体抗体库， 筛选、 表达人源性促甲状腺激素受 体抗体 （TRAb） Fab 段。方法 以人 TSHR 膜外区的氨基端 （hTSHRn）、 人 TSHR 膜外区的羧基端 （hTSHRc） 的融合蛋 白为抗原， 通过数轮 “吸附-洗脱-扩增” 富集噬菌体抗体， 筛选阳性克隆。从已构建的抗体库中筛选到甲状腺刺激性 抗体（TSAb） Fab、 甲状腺阻断性抗体（TBAb） Fab， 噬菌体（Phage） -ELISA 检测选取阳性克隆， PCR 及双酶切鉴定 TRAb 阳性克隆。检测可溶性 TRAbFab 片段阳性克隆的表达， Western blotting 鉴定 TRAb 阳性克隆的免疫学活性， 对 TRAbFab 阳性克隆进行测序分析。结果 经过 5 轮富集筛选， 成功筛选到特异性 TRAb （TSAb、 TBAb） Fab 噬菌体 抗体， 产率分别提高约 77、 94 倍。通过 Phage-ELISA 检测， 鉴定出了具有抗原结合活性的单克隆抗体， 实现可溶性 表达。Western blotting 证实其免疫学活性。测序分析单克隆 48 的轻链可变区与人的免疫球蛋白轻链 Vλ 同源性达 到 94.4%， 重链可变区与人的免疫球蛋白 IgG 重链 VH4 同源性为 88.9%。克隆 56 的轻链可变区与人的免疫球蛋白 轻链 Vλ 同源性达到 95.6%， 重链可变区与人的免疫球蛋白 IgG 重链 VH3 同源性为 84.6%。结论 本研究通过成功 筛选获得人源性 TRAb （TSAb、 TBAb） Fab 片段的单克隆抗体。

关键词: 受体, 促甲状腺素, 抗体, 单克隆, 免疫球蛋白 Fab 片段, 细菌噬菌体, 噬菌体展示库

Abstract: Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6% , and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6% . Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.

Key words: receptors, thyrotropin, antibodies, monoclonal, immunoglobulin Fab fragments, bacteriophages, phage antibody library