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

X. Yanga, R. A. McGraw, X. Su, P. Katakam, W. M. Grosse, O. W. Li, D. C. Ferguson

Research output: Contribution to journalArticlepeer-review

Abstract

The gene encoding the mature β subunit of canine thyroid stimulating hormone (cTSHβ) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSHβ purified from E. coli were generated. The gene fragment that encodes mature TSHβ 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 TSHβ gene and an intron of 450 bp. The two exons, which encode the mature cTSHβ subunit, was joined together by an overlap PCR and was expressed in E. coli as 6XHis- tagged protein. The purified recombinant cTSHβ 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 cTSHβ and subsequently characterized. For transient expression in CHO cells that are permanently transfected with the bovine common α gene, a 60-oligonucleotide signal peptide coding sequence was added to the 5' end of the cTSHβ 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 α and canine TSHβ in heterodimeric confirmation, exhibited TSH bioactivity as indicated by the stimulation of cAMP production in the cultured FRTL-5 thyrocytes. (C) 2000 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)363-378
Number of pages16
JournalDomestic Animal Endocrinology
Volume18
Issue number4
DOIs
StatePublished - May 2000
Externally publishedYes

ASJC Scopus subject areas

  • Food Animals
  • Animal Science and Zoology
  • Endocrinology

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