DETERMINATION OF APHRODISIN GENE OF SYRIAN HAMSTER BY MOLECULAR TECHNIQUE

Abstract

Aphrodisin is a secretory protein expressed in the reproductive organs and parotid salivary gland of the female hamster and acts as an aphrodisiac pheromone. RT-PCR and cloning was applied for the determination of aphrodisin gene from the vaginal tissues of the hamster. In the present study, total RNA was extracted from hamster uterus by Trizol method and apparently undegraded 28S, 18S and 5S species of ribosomal RNA was clearly visible. After performing RT-PCR a distinct band of ~475 bp was obtained and showed in electrophoresis gel analysis which was the partial cDNA of aphrodisin protein. After purification of PCR product and its sequencing using OE-F and OE-R primers confirmed the PCR product was the full-length cDNA of mature aphrodisin of 151 amino acids. The pET 21 plasmid purified from DH5-? cell and ligated with Aph-insert of ~475 bp. Subsequent sequencing confirmed error free ligation and presence of aphrodisin insert. No sequence discrepancy was noted with the published cDNA sequence of mature aphrodisin except for a single base mismatch (GGg for GGA), which did not result in any change in the coded amino acid (glycine). SDS-PAGE analysis showed a major protein greater than 17-kDa was observed in the protein profiles of post-lysis pellet of IPTG induced BL-21 cells but not in their lysates. This 17 kDa protein band should represent recombinant aphrodisin protein which is present in the post-lysis pellet since it is not solubilized by simple sonication. Peptide mass fingerprinting and matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) mass spectra analysis of the trypsin digested ~17 kDa recombinant protein, and mass spectrophotometrical (MS) analysis indicated that the overexpressed ~17 kDa protein was closely similar to aphrodisin of Syrian hamster.

DOI = http://dx.doi.org/10.3329/bjvm.v8i2.11202

Bangl. J. Vet. Med. (2010). 8 (2) : 175-184