Finke R, Seto P, Rapoport B
Thyroid Molecular Biology Laboratory, Veterans Administration Medical Center, San Francisco, California 94121.
J Clin Endocrinol Metab. 1990 Jul;71(1):53-9. doi: 10.1210/jcem-71-1-53.
To define the epitope(s) on human thyroid peroxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease, we constructed and screened a human TPO cDNA sublibrary containing 3.8 million random fragments of human TPO cDNA, each 200-500 basepairs in length. These fragments would code for TPO polypeptides of 66-166 amino acid residues. The validity of this approach was first tested with a murine monoclonal antibody against the denatured human thyroid microsomal antigen (TPO). Analysis of the nucleotide sequence of 14 clones selected from this library enabled molecular identification of the epitope recognized by this monoclonal antibody. In contrast to the data obtained with the monoclonal antibody, sera from patients with Hashimoto's thyroiditis containing polyclonal antimicrosomal/TPO antibodies did not recognize the TPO protein fragments generated by this library. These results differ from previous data obtained with recombinant human TPO fragments generated as bacterial fusion proteins. Our data suggest that, contrary to previous concepts, the natural B-cell epitope(s) on human TPO may be highly conformational (requiring a complex 3-dimensional structure) or may be discontinuous (formed by distant regions of the linear polypeptide chain being brought into apposition by protein folding).
为了确定自身免疫性甲状腺疾病患者血清中抗体所识别的人甲状腺过氧化物酶（TPO）上的表位，我们构建并筛选了一个人TPO cDNA亚文库，该文库包含380万个随机的人TPO cDNA片段，每个片段长度为200 - 500个碱基对。这些片段将编码66 - 166个氨基酸残基的TPO多肽。这种方法的有效性首先用一种针对变性人甲状腺微粒体抗原（TPO）的鼠单克隆抗体进行了测试。对从该文库中选出的14个克隆的核苷酸序列分析，使得能够从分子水平鉴定该单克隆抗体所识别的表位。与用单克隆抗体获得的数据相反，来自桥本甲状腺炎患者的含有多克隆抗微粒体/TPO抗体的血清不能识别该文库产生的TPO蛋白片段。这些结果与先前用作为细菌融合蛋白产生的重组人TPO片段所获得的数据不同。我们的数据表明，与先前的概念相反，人TPO上的天然B细胞表位可能是高度构象性的（需要复杂的三维结构），或者可能是不连续的（由线性多肽链的远距离区域通过蛋白质折叠并列形成）。
