Expression of human thyroid peroxidase in insect cells using recombinant baculovirus.
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)
Kendler
D L
DL
Department of Medicine, Mount Sinai School of Medicine, City University of New York, New York.
Brennan
V
V
Davies
T F
TF
Magnusson
R P
RP
D000818:Animals; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D012831:Bombyx; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007453:Iodide Peroxidase / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013967:Thyroiditis, Autoimmune / Q000097:blood / Q000276:immunology*
D001323:Autoantibodies; D011993:Recombinant Fusion Proteins; C056626:thyroid microsomal antibodies; D004247:DNA; D007453:Iodide Peroxidase