Yanga X, McGraw R A, Su X, Katakam P, Grosse W M, Li O W, Ferguson D C
Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA.
Domest Anim Endocrinol. 2000 May;18(4):363-78. doi: 10.1016/s0739-7240(00)00056-4.
The gene encoding the mature beta subunit of canine thyroid stimulating hormone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSH beta purified from E. coli were generated. The gene fragment that encodes mature TSH beta was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species. The resulting 891 basepairs (bp) of genomic DNA consisted of two coding exons of the canine TSH beta gene and an intron of 450 bp. The two exons, which encode the mature cTSH beta subunit, was joined together by an overlap PCR and was expressed in E. coli as 6xHis-tagged protein. The purified recombinant cTSH beta with a molecular weight of about 15 kDa was recognized by the polyclonal antibodies prepared against the native canine TSH in Western blot. Monoclonal antibodies were raised against the purified cTSH beta and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common alpha gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5' end of the cTSH beta gene before it was cloned into the mammalian expression vector pRSV and used to transfect CHO cells. The medium from these transfected cells, presumably containing the bovine alpha and canine TSH beta in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes.
编码犬促甲状腺激素（cTSHβ）成熟β亚基的基因被克隆、测序，并在大肠杆菌和中国仓鼠卵巢（CHO）细胞中表达，同时制备了针对从大肠杆菌中纯化的重组cTSHβ的单克隆抗体。通过直接聚合酶链反应（PCR），使用基于其他物种共有序列设计的引物，从犬基因组DNA中克隆出编码成熟TSHβ的基因片段。所得的891个碱基对（bp）的基因组DNA由犬TSHβ基因的两个编码外显子和一个450 bp的内含子组成。编码成熟cTSHβ亚基的两个外显子通过重叠PCR连接在一起，并在大肠杆菌中作为6xHis标签蛋白表达。在蛋白质印迹中，分子量约为15 kDa的纯化重组cTSHβ可被针对天然犬TSH制备的多克隆抗体识别。制备了针对纯化的cTSHβ的单克隆抗体，并随后对其进行了表征。为了在永久转染了牛共同α基因的CHO细胞中进行瞬时表达，在将cTSHβ基因克隆到哺乳动物表达载体pRSV中并用于转染CHO细胞之前，在其5'端添加了一个60个寡核苷酸的信号肽编码序列。这些转染细胞的培养基可能含有异源二聚体形式的牛α和犬TSHβ，如在培养的FRTL-5甲状腺细胞中刺激cAMP产生所示，表现出TSH生物活性。
