Studies on Diversity of Anti-TSH Receptor Antibody by Gene Technology.

用抗TSH受体的单抗克隆了插入Lamda GT 11噬菌体的人甲状腺癌基因文库。获得的克隆大小在1020~1900bp之间，经Southern Blott杂交，表达的融合蛋白用于Western Blott分析。Graves病患者和特发性甲状腺功能减退症患者的血清显示融合蛋白呈阳性，而正常人和桥本病患者的血清则不呈阳性。用双脱氧法对其进行核苷酸序列分析。计算机检索表明，该基因与仓鼠的延伸因子2(EF-2)有很强的同源性。分析了TSH受体克隆克隆失败的原因，认为是由于寡核苷酸DT引物合成的cDNA文库和文库长度有限(2kb)所致。另用抗TSH受体的单抗克隆了随机引物法构建的正常人甲状腺组织的cDNA文库。其中一个克隆的cDNA具有约2000kb的阅读框架，与最近发表的TSH受体的核苷酸序列几乎完全一致。在EF-2和TSH受体上发现了两个相同的4个氨基酸序列，这可能是这些蛋白的共同表位。

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.