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当前位置: 首页 > 文章 > 可溶性Sox2-11R重组蛋白的制备与鉴定 安徽农业大学学报 2014,41 (5) 88-94
Position: Home > Articles > Preparation and function analysis of soluble Sox2-11R recombinant protein Journal of Anhui Agricultural University 2014,41 (5) 88-94

可溶性Sox2-11R重组蛋白的制备与鉴定

作  者：
李侠;姜少帅;韩菲;张鹏飞;张运海
单  位：
安徽农业大学动物科技学院;安徽地方畜禽遗传资源保护与生物育种省级实验室
关键词：
Sox2;重组蛋白;可溶性;制备
摘  要：
旨在通过原核表达制备可溶性Sox2-11R重组蛋白,并验证其穿膜能力。首先,采用增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)与蛋白转导域11聚精氨酸(11 arginine,11R)的融合表达和与成纤维细胞的共孵育验证11R的穿膜能力。其次,将目的基因Sox2-11R在原核表达载体pET30a中表达,通过优化表达条件获取可溶性Sox2-11R重组蛋白。最后,利用Ni柱纯化重组蛋白,SDS-PAGE及Western blotting对其进行鉴定;并用免疫荧光染色验证重组蛋白是否具有进入细胞核的能力。结果表明,成功构建了pET30a-Sox2-11R原核表达载体,纯化的Sox2-11R重组蛋白具有穿膜能力,且在细胞培养基中具有较高溶解度,这为制备其他重编程因子的重组蛋白提供了参考,也为外源蛋白诱导iPSCs奠定了基础。
译  名：
Preparation and function analysis of soluble Sox2-11R recombinant protein
作  者：
LI Xia;JIANG Shaoshuai;HAN Fei;ZHANG Pengfei;ZHANG Yunhai;Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Bio-Breeding,School of Animal Science and Technology, Anhui Agricultural University;
关键词：
Sox2;;recombinant protein;;solubility;;preparation
摘  要：
The aim of the study was to prepare soluble recombinant protein Sox2-11 R with cell penetrating peptide using prokaryotic expression system and analyze its functions. Firstly, the cell permeability of the recombinant protein was determined by designing the 11 R protein transduction domain to the C-terminal of EGFP and treated fibroblast cells with the purified protein. Secondly, the soluble protein was obtained by expressing the Sox2-11 R in pET30 a vector and various induction conditions were tested. Finally, the fusion protein was purified by Ni affinity chromatography and identified by SDS-PAGE and Western blotting. The ability of cell permeability was analyzed using immunocytochemistry. The results showed that the vector pET30a-Sox2-11 R was successfully constructed and the purified soluble recombinant protein Sox2-11 R showed the biological activity, efficient cell permeability, and relatively high solubility in the cell medium. This study laid a good foundation for preparation of other reprogramming factors and induction of iPSCs with exogenous proteins.

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