关键词: 比格犬, 雌激素受体β(ER)β, 原核表达

Abstract: Objective To obtain the estrogen receptor β (ERβ) and its splicing isoforms for elucidating its functional roles in the reproductive manipulation of Beagle dogs, the prokaryotic expressed canine pMAL-p5x/ERβ480 was established and optimized. Methods The cDNA specifically encoding ERβwas amplified from the total RNA of the Beagle dog hypothalamus, sequenced and blasted against other ERβcDNA sequences in the GeneBank. The amplified cDNA fragment was then cloned into a prokaryotic expression vector, pMAL-p5x, to produce recombinant plasmid pMAL-p5x/ERβ480. The constructed recombinant plasmid was transformed into E.coli DH α and E.coli BL21(DE3). The MBP-ERβ fusion protein was induced after the addition of IPTG into the growth media. The expressed product was purified by Amylom Resin affinity chromatography, and identified by SDS-PAGE. Results Sequence analysis indicated that the coding region of the cDNA fragment was about a length of 480 bp. The fusion protein was expressed comparatively well under the following conditions : in 0.2% glucose, 100 µg/ml Ampcillin, 0.1mmol/L IPTG at 37℃ for 5h or in 0.2% glucose, 50 µg/mL Ampcillin, 0.2 mmol/L IPTG at 37℃ for 3h. The SDS-PAGE results showed that the molecular weight of recombinant ERβ was of about 60 000, just as expected. Conclusion The pMAL-p5x/ERβ prokaryotic recombinant plasmid was developed successfully, and the beagle dogs MBP-ERβ fusion protein was obtained and biochemical characterized to some extent. It was anticipated that the fusion protein would be useful for the production of the dog species-specific ERâ polyclonal antibody and for the further functional analysis.

Key words: Beagle dogs, Estrogen receptor β(ER)β, Prokaryotic expression