The Expression Of Human Thyroid Peroxidase "Hot Spot" Gene In Escherichia Coli And The Clinical Application

Human Thyroid Peroxidase (hTPO) plays an important role in thyroid hormone biosynthesis. TPO is also a major autoantigen in autoimmune thyroid disease (AITD). It has great value to detect anti-hTPOAb in the diagnosis of AITD (particularly in Hashimoto's thyroiditis).Objective: To use the recombinant extracellular region proteins of hTPO obtained with molecular biological method as the antigen and to establish the ELISA used in the clinical diagnosis and effect monitoring.Methods: Transform the recombinant plasmid pGESJ including hTPO epitopes (gene 1702~2622, encoding A568~874) into E. Coli BL21 and express the recombinant extracellular region proteins with the existing of the inducer. GST-hTPO fusion protein with satisfactory immune activity was obtained after purification. ELISA to detect hTPO autoantibody in patients' sera with GST-hTPO fusion protein as the antigen had been established and applied in the clinical diagnosis and effect monitoring of AITD.Results: The E. Coli BL21 was selected as the expressing strain and IPTG (isopropyl β-D-thoiglactoside) as inducer with the concentration of 0.1 mM. The optimum temperature and time were 25℃ and 5.5 hours. The expressed protein was purified with Glutathione Sepharose 4B by affinity chromatography. The molecular weight of GST-hTPO was 61.24 KD. The producing rate of GST-hTPO fusion protein was 5mg/L medium and its satisfactory antigen activity was verified by ELISA. There was no cross-reaction between GST-hTPO fusion protein and Tg (Thyroglobulin). The definition of the optimum condition of ELISA: The optimum antigen coating dose was 1 μg/well. The dilution power of patients' sera and antiantibody were 1:1 and 1:20000 respectively;The optimum timeto show color was 37°C and 45minutes. The repetition of ELISA: The precision for the assay was 5. 51%^P 6. 75% CV inside the batch and 8. 95 ^fl 9. 51% among the batches. Clinical application: The anti-hTPO test showed positive results in 79% of Hashimoto's thyroiditis and 10.4% in Graves' disease. The results all showed negative in simple goiter and subacute thyroiditis patients.The results of Hashimoto's thyroiditis patients showed significant difference compared with other groups(p< 0.05).Conclusion: ELISA method using the gene cloning product— GST-hTPO fusion protein as the antigen provided useful tools for determination of autoantibodies in primary diagnosis and evaluation of the treatment effect as shown with AITD. The high precision of the assay made it perfectly suited to routine diagnostic use.