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 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.
Yanga
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Department of Physiology and Pharmacology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602, USA. yang3@niehs.nih.gov
McGraw
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Su
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Katakam
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Grosse
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Li
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Ferguson
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001483:Base Sequence; D015153:Blotting, Western / Q000662:veterinary; D016466:CHO Cells; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017421:Sequence Analysis; D012689:Sequence Homology, Nucleic Acid; D013972:Thyrotropin / Q000737:chemistry / Q000235:genetics*; D014162:Transfection / Q000235:genetics
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011994:Recombinant Proteins; D013972:Thyrotropin; D004247:DNA