Expression of Recombinant Human Thyroid Peroxidase by the Baculovirus System and Its Use in Elisa Screening for Diagnosis of Autoimmune Thyroid Disease
作者:
HaubruckHeinz,
MauchLudwig,
CookNeil J.,
SteffensUte,
HuntNicholas,
BertholdHeike,
NiemannHeike,
WirbelauerChristiane,
NorthemannWolfgang,
期刊:
Autoimmunity
(Taylor Available online 1993)
卷期:
Volume 15,
issue 4
页码: 275-284
ISSN:0891-6934
年代: 1993
DOI:10.3109/08916939309115749
出版商: Taylor&Francis
关键词: thyroid peroxidase;autoimmunity;Hashimoto's thyroiditis;Graves' disease;recombinant;expression;baculovirus
数据来源: Taylor
摘要:
The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection ofSpodoptera frugiperda(Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by convential methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n= 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.
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