实验动物与比较医学2013，Vol.33Issue(3)：174-179,6.DOI:10.3969/j.issn.1674-5817.2013.03.002
比格犬雌激素受体β的原核表达、纯化及鉴定
Prokaryotic Expression, Purification and Identification of Estrogen Receptor in Beagle Dogs
摘要
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 ERβ was amplified from the total RNA of the Beagle dog hypothalamus,sequenced and blasted against other ERβ cDNA 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 5α 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 ER(a) polyclonal antibody and for the further functional analysis.关键词
比格犬/雌激素受体β(ERβ)/原核表达Key words
Beagle dogs/ Estrogen receptor β(ERβ)/ Prokaryotic expression分类
生物科学引用本文复制引用
许琴,赵彦斌,任秀梅,孙兆增,叶华虎,刘一,李微,白杰英,曾林..比格犬雌激素受体β的原核表达、纯化及鉴定[J].实验动物与比较医学,2013,33(3):174-179,6.基金项目
国家自然科学基金资助项目(31172164) （31172164）
