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

用抗TSH受体单克隆抗体克隆了人甲状腺癌λ gt 11噬菌体cDNA文库。获得大小为1020 ~ 1900 bp的cDNA克隆，经Southern blotting杂交，IPTG诱导表达的融合蛋白用于Western blotting分析。Graves病和特发性甲状腺功能减退症患者的血清显示融合蛋白的阳性染色，而正常人或桥本病患者的血清则没有。用双脱氧法分析其核苷酸序列。计算机检索表明，该cDNA与金黄地鼠的延伸因子2（EF-2）有很强的同源性。考虑到用寡聚dT引物合成的cDNA文库及cDNA文库的长度（2kb）限制性，不能成功克隆TSH受体克隆，用抗TSH受体单克隆抗体，用随机引物法制备了正常人甲状腺组织cDNA文库。其中一个cDNA的阅读框约为2000 Kb，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受体中发现了两个相同的4个氨基酸序列，可能是这两种蛋白的共同表位。

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.