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 β in Beagle Dogs

作　　者：许琴[1,2] 赵彦斌[1] 任秀梅[1,3] 孙兆增[1] 叶华虎[1] 刘一[1] 李微[1] 白杰英[1] 曾林[1] 胡仲明[1] 

机构地区：[1]军事医学科学院实验动物中心,北京100071 [2]兰州军区乌鲁木齐总医院动物实验科,乌鲁木齐830000 [3]吉林大学畜牧兽医学院,长春130062

出　　处：《实验动物与比较医学》2013年第3期174-179,共6页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金资助项目（31172164）

摘　　要：目的研究比格犬雌激素受体β（Estrogenreceptorβ，ERβ）及其剪接异构体在生殖调控中的功能，构建比格犬ERβ的pMAL-p5x／ERβ 480DE3E．coli重组菌株，并进行纯化和鉴定。方法Trizol法提取发情期比格犬下丘脑总RNA，RT-PCR获得cDNA，以NCBI网站公布的比格犬ERβ基因CDS序列设计特异性引物，扩增ERβ保守区基因编码序列（16-496bp），连接原核表达载体pMAL-p5x，筛选、鉴定阳性克隆并测序。将重组质粒转化至大肠杆菌DH5α，再转化大肠杆菌BL21（DE3），用IPTG诱导表达，表达产物经经麦芽糖亲和树脂（AmylomResin）亲和层析分离纯化，并对纯化的融合蛋白进行鉴定。结果构建了pMAL-p5x／ERa480重组质粒，在DE3大肠杆菌中诱导表达出MBP-ERβ融合蛋白，SDS-PAGE显示分子量约为60000，与预期结果一致：优化了MBP-ERβ表达体系的表达条件：分别在0．2％葡萄糖，1000g／mlAmpcillin，0．1mmol／LIPTG37℃培养5h或在0．2％葡萄糖，50μg／mlAmpcillin，0．2mmol／LIPTG37℃培养3h，融合蛋白表达效果比较好。结论构建了pMAL-p5x／ERβ480原核表达重组质粒，获得了比格犬MBP—ERB融合蛋白，为犬种属特异性ERB多克隆抗体的制备及功能分析奠定了基础。Objective To obtain the estrogen receptor β （ERβ） and its splicing isoforms for elucidat- ing 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 prokary- otic 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, 100μg/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 produc- tion of the dog species-specific ERa 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 

分 类 号：Q95-33[生物学—动物学]