| 离子通道相关蛋白FXYD6功能区的原核表达及纯化 |
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| 引用本文： | 陈雄飞,周宁新. 离子通道相关蛋白FXYD6功能区的原核表达及纯化[J]. 山东医药, 2010, 50(51): 4-6 |
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| 作者姓名： | 陈雄飞 周宁新 |
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| 作者单位： | 辽宁医学院;中国人民解放军第二炮兵总医院; |
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| 基金项目： | 国家自然科学基金资助项目 |
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| 摘 要： | 目的构建FXYD6蛋白功能区（FXYD6-ex）的原核表达质粒,并诱导FXYD6-ex在原核细胞中表达与纯化,以进一步研究FXYD6功能。方法用全长FXYD6 cDNA扩增FXYD6-ex,扩增产物插入克隆质粒载体pMD18-T Simple并用限制性内切酶酶切,将目的片段插入以限制性内切酶切后的表达质粒载体pET28a（＋）中,经PCR电泳和测序证实后转化宿主大肠杆菌Rossetta,异丙基-β-8-D硫代半乳糖（IPTG）诱导重组蛋白表达,镍柱纯化重组蛋白,十二烷基硫酸钠—聚丙烯酰胺（SDS-PAGE）凝胶和Western blot检测鉴定蛋白表达及纯化情况。结果成功构建pET28a（＋）-FXYD6-ex大肠杆菌表达载体,并实现该蛋白在大肠杆菌中的高表达,分离纯化的产物纯度达90%。结论本研究成功对FXYD6-ex进行原核表达及纯化;此为后续制备该蛋白抗体库及进一步研究其具体功能奠定了良好基础。
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| 关 键 词： | FXYD6 蛋白表达 蛋白纯化 |
Prokaryotic expression and purification of functional domain of Ion channel protein FXYD6 |
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| CHEN Xiong-fei,ZHOU Ning-xin. Prokaryotic expression and purification of functional domain of Ion channel protein FXYD6[J]. Shandong Medical Journal, 2010, 50(51): 4-6 |
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| Authors: | CHEN Xiong-fei ZHOU Ning-xin |
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| Affiliation: | CHEN Xiong-fei1,ZHOU Ning-xin(1 Liaoning Medical College,Jinzhou 121001,P.R.China) |
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| Abstract: | Objective To contribute expression plasmid of fuctional domain of FXYD6(FXYD6-ex) and induce its expression and purification in prok-aryocyte.Methods T he DNA of FXYD6-ex was amplified by PCR with FXYD6 cDNA,and then inserted into cloning vector pMD18-T Simple.The recombinant was digested by restriction endon uclease,the interest fragment was inserted into expression vector pET28a(+)dige sted by the same restriction endonuclease.Confirmed by PCR electrophoresis and sequence analysis,the verified recombinant... |
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| Keywords: | FXYD domain containing ion transport regulator 6 protein expression p rotein purification |
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