Expression of human thyroid peroxidase in insect cells using recombinant baculovirus
| Autor: | Terry F. Davies, Veronica Brennan, David L. Kendler, Ronald P. Magnusson |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Antigenicity
Recombinant Fusion Proteins Microgram Blotting Western Genetic Vectors Enzyme-Linked Immunosorbent Assay CHO Cells Recombinant virus Iodide Peroxidase Biochemistry law.invention Sepharose Endocrinology Antigen Thyroid peroxidase law Cricetinae Animals Humans Molecular Biology Cells Cultured Autoantibodies biology Chinese hamster ovary cell Thyroiditis Autoimmune DNA Bombyx Virology Molecular biology biology.protein Recombinant DNA Electrophoresis Polyacrylamide Gel Baculoviridae |
| Zdroj: | Molecular and Cellular Endocrinology. 93:199-206 |
| ISSN: | 0303-7207 |
| DOI: | 10.1016/0303-7207(93)90124-3 |
| Popis: | The baculovirus expression system was used to overexpress recombinant human thyroid peroxidase. Sf-9 cells infected with the recombinant virus AcMNPV-hTPO synthesized hTPO protein (hTPO-bac) immunogenic on Western blots when probed with either rabbit anti-TPO peptide sera or pooled human anti-TPO sera (MS12/89). hTPO-bac was a major constituent of the membrane fraction from the infected cells, constituting 14.9% and 10.1% of the 1% deoxycholate-soluble and insoluble fractions, respectively, as judged by densitometry. Recombinant hTPO-bac was extracted from cellular membranes with 1% deoxycholate and partially purified by Sepharose 6B column chromatography. Specific immunoreactivity of MS12/89 to hTPO-bac on microtiter plates was seen using ELISA. Detergent extract from wild-type virus-infected Sf-9 cells was used as background control antigen; no specific reactivity to either hTPO-bac or control antigen was seen with control sera. To determine antigenic potency, MS12/89 was incubated with increasing concentrations of various preparations of hTPO antigen and with ovalbumin as control. The capacity of the partially purified hTPO-bac to immunoneutralize human anti-hTPO standard at 50% inhibition of binding was 0.01 U/microgram hTPO-bac (NIBSC Units), compared with 0.5 U/microgram and 0.06 U/microgram for natural hTPO and CHO-hTPO, respectively. When ELISA was performed using clinical samples of human sera to detect hTPO autoantibodies, results using hTPO-bac correlated well with those using hTPO from Graves' thyroid tissue (r = 0.85, p = 0.02) and those using recombinant hTPO from Chinese hamster ovary cells (hTPO-CHO) (r = 0.85, p = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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