人甲状腺过氧化物酶抗原决定簇基因在大肠杆菌的表达及临床测定应用

The expression of human thyroid peroxidase epitope gene in Escherichia coli and its clinical assay application

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 ;

Objective To express human thyroid peroxidase (hTPO) epitopes gene and apply its products in clinical assay. Methods hTPO epitopes gene was cloned into expression vector pGEX 4T 3 then transformed into E. coli BL21. Expression of hTPO gene was induced by isopropyl β D thiogalactoside, expressed product (GST hTPO) was purified by affinity chromatography and their immunoactivity was demonstrated. ELISA technique using GST hTPO as antigen was established for determining TPOAb. Serum TPOAb level was determined, and HLA DR antigen, dendritic cells and lymphocytes in the thyroid gland tissue were observed in these same AITD patients.Results GST hTPO acquired from procaryotic expression had high purity and good immunoactivity. The CVs of the ELISA technique established with GST hTPO were between 5.93%～7.59%. A significantly positive correlation was found between the TPOAb levels determined respectively by ELISA and RIA method. Serum TPOAb level and distribution of HLA DR antigen and dendritic cells showed the same ascending tendency following the aggravation of lymphocyte infiltration. Conclusion Product of genetic engineering, GSH hTPO, can be used to establish a clinical assay for TPOAb. The correlation between the level of serum TPOAb and the detriment of thyroid tissue is demonstrated.