Nucleotide sequence of equine erythropoietin and characterization of region-specific antibodies.
OBJECTIVE
To determine the full-length complementary DNA (cDNA) sequence of equine erythropoietin (EPO) and to develop region-specific antibodies to differentiate equine EPO (eEPO) and human EPO (hEPO).


METHODS
RNA and lysate extracted from renal tissues of an adult Thoroughbred.


METHODS
Full-length cDNA was determined by use of a reverse transcriptase-polymerase chain reaction assay and a rapid amplification of cDNA ends method. The deduced amino acid sequence was compared with sequences of EPO reported for other species. Furthermore, 4 synthetic peptides were designed in 2 distinctive parts of the eEPO and hEPO amino acid sequences to obtain antibodies specific for eEPO and hEPO. Specificity of the antibodies was tested against supernatant of homogenized equine kidney and recombinant hEPO (rhEPO) by use of western immunoblotting techniques.


RESULTS
Analysis of the 1,181 bp in the nucleotide sequence revealed that eEPO was a residue of 192 amino acids. Similarity of eEPO with amino acid sequences of EPO from other species was 81.0% to 90.6%. Antibodies were specifically recognized by eEPO or rhEPO molecules. Anti-hEPO (161 to 165) antibody specifically recognized rhEPO. In contrast, anti-eEPO (133 to 144) antibody reacted with the equine kidney lysate.


CONCLUSIONS
We determined the cDNA and amino acid sequence of eEPO and developed region-specific antibodies that specifically recognized eEPO or rhEPO. These antibodies may be useful in distinguishing rhEPO from eEPO in a test to detect the misuse of rhEPO in racehorses.
Sato
Fumio
F
Equine Research Institute, Japan Racing Association, 321-4 Tokami, Utsunomiya 320-0856, Japan.
Yamashita
Shozo
S
Kugo
Takaharu
T
Hasegawa
Telhisa
T
Mitsui
Isao
I
Kijima-Suda
Isao
I
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics*; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics* / Q000276:immunology; D006736:Horses / Q000235:genetics*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis; D017422:Sequence Analysis, DNA
D000906:Antibodies; D018076:DNA, Complementary; D004921:Erythropoietin