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目的 表达有抗原活性的人甲状腺过氧化物酶 (hTPO)片段 ,并将表达产物用于临床测试。方法 将hTPO抗原决定簇基因连入 pGEX 4T 3,然后转入E .coliBL2 1,异丙基 β D 硫代半乳糖苷诱导表达。表达产物谷胱甘肽巯基转移酶 (GST) hTPO经亲和层析纯化 ,并鉴定免疫学活性。以GST hTPO为抗原建立检测血清TPO抗体 (Ab)的ELISA方法。用此方法检测一批自身免疫性甲状腺疾病 (AITD)患者血清TPOAb ,用病理学方法观察同批患者甲状腺组织中HLA DR抗原、树突状细胞及淋巴细胞浸润程度。结果 成功地表达了hTPO抗原决定簇基因 ,纯化产物GST hTPO纯度高 ,有良好的免疫学活性 ,以此抗原建立的ELISA方法有良好的重复性 ,CV在 5 .93 %～ 7.5 9%之间。ELISA方法与RIA方法比较 ,检测结果显著相关 (n =37,r =0 .6 13,P <0 .0 0 1)。血清TPOAb水平及HLA DR抗原、树突状细胞分布随甲状腺组织淋巴细胞浸润程度加重呈逐渐增多趋势。结论 原核表达产物GST hTPO可用于建立TPOAb常规检测方法 ;AITD患者血清TPOAb水平与甲状腺组织损伤有密切关系
Objective To express human thyroid peroxidase (hTPO) fragments with antigenic activity and to use the expressed products for clinical testing. Methods The hTPO antigenic determinant gene was ligated into pGEX 4T 3 and then transformed into E.coli BL21. The induced expression was induced by isopropyl β D thiogalactoside. The expression product glutathione S-transferase (GST) hTPO was purified by affinity chromatography and immunological activity was identified. An ELISA method for detecting serum TPO antibody (Ab) was established using GST hTPO as antigen. This method was used to detect serum TPOAb in patients with autoimmune thyroid disease (AITD) and to observe the degree of HLA DR antigen, dendritic cells and lymphocyte infiltration in the thyroid tissues of the same patients by pathology. Results The gene of hTPO epitope was successfully expressed. The purified product GST hTPO was of high purity and good immunological activity. The ELISA method established by this antigen showed good reproducibility. The CV was between 5.93% and 7.59% . The results of ELISA were significantly correlated with those of RIA (n = 37, r = 0.61, P <0.01). Serum levels of TPOAb and HLA DR antigen, dendritic cells distribution with thyroid lymphocyte infiltration degree increased gradually increased. Conclusion The prokaryotic expression product GST hTPO can be used to establish the routine detection of TPOAb; serum TPOAb levels in patients with AITD and thyroid tissue damage are closely related