Rayalam S., Eizenstat L.D., Hoenig M., et al.
Conference Proceedings, (2005). American College of Veterinary Internal Medicine, Baltimore:
Hyperthyroidism is the most common endocrine disorder of elderly
cats. However, its early diagnosis and studies of risk factors have
been hampered by the inavailability of a feline-specific TSH assay.
Although there have been attempts to use available canine TSH
immunoassays in cat sera, cross-reactivity is insufficient for
distinguishing suppressed values from normal. Feline-specific
peptide and antibody reagents are critical for development of a
clinically useful immunoassay. In the current study, the genes
encoding the mature common pituitary alpha and hormone-specific
beta subunits of feline thyroid stimulating hormone (fTSH) were
cloned and sequenced. The feline common pituitary alpha gene was
cloned from the total RNA extracted from the feline pituitary gland
by the reverse transcription polymerase chain reaction (RT-PCR).
The gene fragment that encodes mature TSHbeta was cloned from the
feline genomic DNA by direct polymerase chain reaction (PCR).
Following the experience with improved expression of the human
TSH beta subunit, the second intron was included to produce a
fTSHbeta mini-gene construct. For both subunits, primers were based
on consensus sequences from TSH in other species. The resulting 510
bp PCR product for the alpha subunit included the full coding
sequence of the 96 amino acid mature subunit preceded by that of a
24 amino acid signal peptide. The predicted amino acid sequence of
the mature α subunit had the following species homologies: to canine
(98%), bovine (95%), tiger (97%) and human (69%). The 850 bp
sequence of fTSHbeta genomic DNA consisted of two coding exons,
an intron of 418 bp and a 60 bp signal sequence. The mature
fTSHbeta subunit is homologous to canine (94%), human (88%),
bovine (91%) and equine (95%) TSHbeta subunits. An
immunoaffinity tag FLAG was added to 3’ end of the alpha gene to
facilitate detection by Western blot and purification. In human
pituitary glycoproteins, single chain or yoked analogues have been
shown to have increased stability and bioactivity. In the current
study, yoked fTSH (yfTSH) was developed by fusing the nucleotides
encoding the C-terminus of the beta subunit to the N-terminus of the
alpha subunit by using DNA encoding the C-terminal peptide (CTP)
of human chorionic gonadotropin beta subunit of 26 amino acids as a
linker peptide. The yoked fTSH construct encoded from N-terminus
to C-terminus: beta-CTP-alpha-FLAG. The construct of 1260 bp was
cloned and sequence confirmed. This work describes for the first time
the full coding sequences of the two subunits of fTSH and has
produced DNA constructs for its in vitro expression and
purification.