| Project/Area Number |
63570546
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内分泌・代謝学
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| Research Institution | Keio University |
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Principal Investigator |
ICHIKAWA Yoichi Keio Univ. Dept. of Medicine, Assistant Prof,, 医学部, 講師 (30051402)
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| Co-Investigator(Kenkyū-buntansha) |
AKAMA Hideto Keio Univ. Dept. of Medicine, Assistant., 医学部, 助手 (10202524)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | TSH / TSH receptor / cDNA / EF-2 / Graves' disease / monoclonal antibody / idiopathic hypothyroidism / TSH受容体 / 抗TSH受容体抗体 / 遺伝子工学 / クローニング / 自己抗体 |
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| Research Abstract |
A cDNA library made of human thyroid carcinoma inserted to lamda gt 11 bacteriophage was cloned with anti-TSH receptor monoclonal antibodies. The cDNA clones obtained had the size of 1020 to 1900 bp and hybridized each other on Southern blott analysis.
The fusion protein expressed with IPTG was used for the Western blott analysis. Sera of patients with Graves' disease or those of idiopathic hypothyroidism showed positive staining of the fusion protein, whereas the sera of normal subjects or patients with Hashimoto's disease did not. The cDNA was analyzed for the nucleotide sequence by dideoxy method. Computer search revealed that the cDNA showed strong homology with elongation factor 2(EF-2) of hamster. The reasons of unsuccessful cloning of TSH receptor clone were considered due to cDNA library synthesized with olige dT primer and the limited length (2 kb) of cDNA library.
Another cDNA library made of normal human thyroid tissue with ramdom primer method was cloned with anti-TSH receptor monoclonal anti bodies. One of the cloned cDNA had the reading frame of about 2000 Kb, which was almost completely coincided with the nucleotide sequence of TSH receptor published recently. Two identical 4 amino acids sequences were found in EF-2 and TSH receptor, which might be the common epitopes of these proteins.
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