Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells

The gene encoding the mature beta subunit of canine thyroid stimulating hor mone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli an d in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against t he recombinant cTSH beta purified from E. coli were generated. The gene fra gment that encodes mature TSH beta was cloned from the canine genomic DNA b y direct polymerase chain reaction (PCR) using primers that were designed b ased on the consensus sequences from other species. The resulting 891 basep airs (bp) of genomic DNA consisted of two coding exons of the canine TSH be ta gene and an intron of 450 bp. The two exons, which encode the mature cTS H 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 antibodie s prepared against the native canine TSH in Western blot. Monoclonal antibo dies were raised against the purified cTSH beta and subsequently characteri zed. For transient expression in CHO cells; that are permanently transfecte d with the bovine common alpha gene, a 60-oligonucleotide signal peptide co ding sequence was added to the 5' end of the cTSH beta gene before it was c loned into the mammalian expression vector pRSV and used to transfect CHO c ells. The medium from these transfected cells, presumably containing the bo vine ct and canine TSH beta in heterodimeric confirmation, exhibited TSH bi oactivity as indicated by the stimulation of cAMP production in the culture d FRTL-5 thyrocytes. (C) 2000 Elsevier Science Inc. All rights reserved.