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Determination of Aphrodisin Gene of Syrian Hamster by Molecular Technique

M. Z. I. Khan
Department of Anatomy and Histology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

P. K. De
Centre for Cellular & Molecular Biology, Uppal Road, Hyderabad 500007, Andhra Pradesh, India

Abstract/ Executive Summary:

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-α 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 (MALDITOF) 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. Key words:

Keywords: Syrian hamster, Aphrodisin protein, Overexpression

Location: Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Commodity: Rats

Objectives:

To be done using the recombinant aphrodisin protein, which will not have any bound pheromonal ligand of hamster origin, and
To obtain recombinant aphrodisin protein.

Methodology:

The study was carried out in the Centre for Cellular and Molecular Biology, Hyderabad, India. In the present study the Syrian (golden) female hamsters (Mesocricetus auratus) were bred and maintained in the institutional animal facility. They were reared in a case with food and water ad libitum. Upper vagina and utero-vaginal junction were taken and total RNA was extracted by Trizol(R) reagent (GIBCO BRL, Grand Island, NY, USA) based on the procedure. In brief 100 mg of vaginal tissue close to the external os was dissected out with sharp surgical instrument and immediately homogenized (Kinematica GmbH homogenizer, Switzerland) in a Sorvall tube containing 1 ml of TRIzol Reagent (a monophasic solution of phenol and guanidine isothiocyanate). Following homogenization insoluble materials was removed from the homogenate by centrifugation (Eppendorf centrifuge 5810 R) at 12,000 x g for 10 minutes at 4⁰C. Transferred the clear homogenates to a fresh centrifuge tube (1.5 ml tube) and incubated it for 5 minutes at 15–30⁰C to permit complete dissociation of nucleoprotein complexes. Added 0.2 ml of chloroform per 1 ml of Trizol reagent for phase separation. Cap microfuge tube to secure. Shake the tube vigorously by hand for 15 seconds and incubate at room temperature for 2–3 minutes. Centrifuged the sample at 12,000 x g for 15 minutes at 2-8⁰C. The cloned plasmid then transformed into BL-21 cells and inducted with IPTG (isopropyl β-D thiogalacto pyranoside) for over-expression of aphrodisin which then sonicated and run through SDS-PAGE gels at 7mA constant current. The gel was removed and stained in 0.25% coomassie blue dye (made in 45% Methanol, 45% water and 10% acetic acid) for 3-4 hrs. Destaining was done in 45 methanol, 45% water and 10% acetic acid mixture. Matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) mass spectra were recorded in a Voyager-DE STR mass spectrometer (Applied Biosystem) to check the homogeneity of the purified protein and to determine their masses.

Source of Information: Bangl. J. Vet. Med. (2010). 8(2) : 175 – 184

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the present study, total RNA was extracted from hamster uterus by Trizol method and apparently un degraded 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-α 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 (MALDITOF) 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.

Knowledge Management: Journal