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| 重组釉原蛋白的提取和纯化 | |
| 引用本文： | 孙樱林,冯海兰,黄薇.重组釉原蛋白的提取和纯化[J].口腔颌面修复学杂志,2010,11(2). |
| 作者姓名： | 孙樱林 冯海兰 黄薇 |
| 作者单位： | 1. 首都医科大学附属北京口腔医院修复科 2. 北京大学口腔医学院修复科,北京,100081 3. 北京大学医学部心血管研究所基因转移与基因治疗实验室,北京,100083 |
| 基金项目： | 国家自然科学基金，教育部高等学校博士学科点专项科研基金 |
| 摘 要： | 目的:通过固相化金属亲和层析的方法,获得较高纯度的具有生物活性的重组釉原蛋白。方法:将重组表达质粒pcDNA3.1TM/Am/myc-His(-)B转染至HEK293A细胞中,用G418持续筛选,培养扩增。收集细胞,加入RIPA细胞裂解液,粗提蛋白。通过镍金属螯合层析的方法,纯化目的蛋白,并进行SDS-PAGE和Westernblot检测,检测蛋白纯化程度和产率。将釉原蛋白冻干,-80℃冻存。结果:SDS-PAGE及WesternBlot检测结果表明,蛋白粗提液中有大小约50KDa的重组釉原蛋白表达,经过HisTrapHPcolumn层析,目的蛋白形成了单一条带,纯化率为91.3%。结论:本实验通过固相化金属亲和层析的方法,获得了高纯度的重组釉原蛋白,为进一步研究蛋白质功能奠定了基础。 |
| 关 键 词： | 釉原蛋白 真核表达 蛋白质纯化 |
Extraction and purified of recombinant human amelogenin |
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| SUN Ying-lin,FENG Hai-lan,HUANG Wei.Extraction and purified of recombinant human amelogenin[J].Chinese Journal of Prosthodontics,2010,11(2). | |
| Authors: | SUN Ying-lin FENG Hai-lan HUANG Wei |
| Institution: | SUN Ying-lin; FENG Hai-lan; HUANG Wei. (Department of Prosthodontics; Peking University School and Hospital of Stomatology; Beijing 100081; China); |
| Abstract: | Objective:The aim of this study was to obtain recombinant human amelogenin with high purity through immobilized metal affinity chromatography (IMAC). Methods: The recombinant expression plasmid pcDNA3.1TM/Am/ myc-His(-)B was transferred into the HEK 293A eukaryocyte cells. The cells were cultured selectively with G418. RIPA cell lysis was used to extract protein primarily. HisTrap HP column was used to extract the recombinant amelogenin with high purity. Then SDS-PAGE and Western Blot were performed to chec... |
| Keywords: | smelogenin eukaryotic expression protein purification |
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