Arscott P L, Koenig R J, Kaplan M M, Glick G D, Baker J R
Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 1996 Mar 1;271(9):4966-73. doi: 10.1074/jbc.271.9.4966.
To define the autoantibody epitopes in amino acids 513-633 of thyroid peroxidase (TPO), a region frequently recognized in thyroiditis, cDNA sequences coding for peptide fragments of this region were amplified and ligated into pMalcRI and pGEX vectors for expression as recombinant fusion proteins. Western blots and enzyme-linked immunosorbent assay were then used to examine the reactivity in sera from 45 Hashimoto's and 47 Graves' disease patients. Two autoantibody epitopes within TPO amino acids 589-633 were identified; 16 of 35 patients reactive to TPO513-633 recognized the epitope of TPO592-613, while 6 patients recognized the epitope of TPO607-633. Eleven other patients with thyroiditis and two with Graves' disease recognized only the whole 589-633 fragment, and this response accounted for the Hashimoto's disease specificity. An amino acid sequence comparison of TPO592-613 with analogous regions of other peroxidase enzymes revealed significant differences in this area, and the substitution of even a single amino acid in one of the epitopes markedly decreased the binding affinity of autoantibodies. Additionally, the exclusive recognition by patients of only one of the epitopes within this region suggests a genetic restriction of the autoantibody response.
为了确定甲状腺过氧化物酶（TPO）513 - 633氨基酸区域中的自身抗体表位，该区域在甲状腺炎中常被识别，编码此区域肽片段的cDNA序列被扩增，并连接到pMalcRI和pGEX载体中，以表达重组融合蛋白。然后使用蛋白质免疫印迹法和酶联免疫吸附测定法检测45例桥本氏病患者和47例格雷夫斯病患者血清中的反应性。在TPO的589 - 633氨基酸区域内鉴定出两个自身抗体表位；在35例对TPO513 - 633有反应的患者中，有16例识别TPO592 - 613的表位，而6例患者识别TPO607 - 633的表位。另外11例甲状腺炎患者和2例格雷夫斯病患者仅识别完整的589 - 633片段，这种反应构成了桥本氏病的特异性。TPO592 - 613与其他过氧化物酶类似区域的氨基酸序列比较显示该区域存在显著差异，并且其中一个表位中即使单个氨基酸的替换也会显著降低自身抗体的结合亲和力。此外，患者仅对该区域内一个表位的特异性识别表明自身抗体反应存在遗传限制。
