Production of recombinant autoantibodies

重组自身抗体的生产

• 批准号: 08557150
• 负责人: AKAMIZU Takashi
• 金额: $ 4.1万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557150/
• 关键词: Immunoglobuilin gene; TSH receptor; anti-TSH receptor antibody; Graves*f disease; recombinant antibody; Immunoglobuilin gene; TSH receptor; anti-TSH receptor antibody; Graves*f disease; recombinant antibody

[Purpose] 1)Anti-thyrotropin receptor autoantibodies are known to be involved in Graves' disease. To elucidate the molecular mechanism of the pathogenesis of Graves' disease, we previously isolated and reconstituted the immunoglobulin genes of two B cell clones producing a monoclonal thyroid stimulating antibody (TSAb), a stimulating type of TSHRAb, obtained from patients with Graves' disease. In the present study, we produced a large amount of recombinant monoclonal TSAbs in eukariotic cells using these genes and applied them to several further investigations 2) We made a DNA construct for transgenic mouse bearing anti-TSH .receptor antibody genes.[Method] 1) We tried to identify their epitopes in the TSHR by using a panel of mutants of the extracellular domain of TSHR.Purified antibodies were radiolabeled and tested for binding to cells expressing high levels of TSHR.2) We tried to isolate heavy and light chain genes containing their promotors and 3'-enhancers from genome DNAs of B c … More ell clones producing monoclonal anti-TSH receptor antibodies by the PCR amplification system. 3) We fried to identify dinucleotide repeat polymorphism near the TSHR gene and performed association studies between genetic markers and Japanese patients with autoimmune thyroid disease (AITD).[Results] 1) Mutations in the N-terminal but not C-terminal region of the extracellular domain of TSHR abrogated or reduced TSAb activities of both antibodies, while they had opposite effects on TSH activity. Although their affinities were lower than that of TSH, their bindings were not displaced by TSH.The antibody bindings were not mutually competitive, either. 2) We isolated heavy and light chain genes containing their promotors and 3'-enhancers from genome DNAs of B cell clones producing monoclonal anti-TSH receptor antibodies by the PCR amplification system. They were legated in tandem.[Conclusion] 1) These findings indicate that these antibodies interact with the multiple conformational arrays of receptor determinants in the N-terminus and transduce a signal through binding sites different from TSH.2) A DNA construct for transgenic mouse bearing anti-TSH receptor antibody genes was preliminary prepared. Less

[目的] 1）已知抗硫代性受体自身抗体参与坟墓疾病。为了阐明坟墓疾病的发病机理的分子机制，我们先前分离并重构了两个B细胞克隆的免疫球蛋白基因，产生了单克隆甲状腺甲状腺刺激抗体（TSAB），这是一种刺激的TSHRAB类型的TSHRAB。在本研究中，我们使用这些基因生产了大量的真主核细胞中的重组单克隆TsAbs，并将它们应用于几项进一步的研究2）我们为转基因小鼠抗体抗体抗体基因制造了DNA构造。对抗体进行放射标记并测试与表达高水平TSHR的细胞结合。2）我们试图通过PCR增强系统将含有其启动子的重型和轻链基因与B C C的基因组DNA分离，这些基因组DNA与B C的基因组DNA相结合。 3）我们油炸以鉴定TSHR基因附近的二核苷酸重复多态性，并在遗传标记和日本患有自身免疫性甲状腺疾病（AITD）的日本患者之间进行了关联研究。[结果] 1）在TSHR胞外影响或减少TSAB活动的tshr胞外域的N末端但没有C-C-末端区域的突变。尽管它们的亲和力低于TSH的亲和力，但它们的结合并未被TSH置换。抗体结合也不相互竞争。 2）我们从B细胞克隆的基因组DNA中分离了包含其启动子和3'-增强剂的重链基因，这些基因组DNA通过PCR扩增系统产生单克隆抗TSH受体抗体。 [结论] 1）这些发现表明，这些抗体与N末端中的受体确定剂的多构象阵列相互作用，并通过与TSH的结合位点进行了与TSH的结合位点的信号。较少的