带有穿膜肽重组PTD-Sox2的制备及活性鉴定  

Preparation and Activity Identification of Recombinant PTD-Sox2

作　　者：刘晓飞[1] 余榕捷[1] 王静静[1] 苏航[2] 黄霖[1] 陈建苏[2] 

机构地区：[1]暨南大学生物工程研究所,广东广州510632 [2]暨南大学医学院眼科,广东广州510632

出　　处：《生物技术》2011年第3期23-29,共7页Biotechnology

基　　金：国家自然科学基金项目资助(No.30973244);Supported by the National Natural Science Foundation of China(No.30973244)

摘　　要：目的:制备带有穿膜肽的重组蛋白PTD-Sox2并对其活性进行鉴定。方法:通过将Sox2基因与穿膜肽基因PTD融合,利用原核表达载体pKYB进行表达,用Ni柱制备、纯化融合蛋白;用Western blot对其进行鉴定;用FITC标记PTD-Sox2,与CHO细胞孵育,检测其穿膜能力;用FRET法检测转录因子Sox2结合目的DNA的活性。结果:构建了带有穿膜肽重组PTD-Sox2的原核表达菌,制备并纯化PTD-Sox2,对其活性鉴定。结论:PTD-Sox2能够穿过细胞膜与核膜,进入细胞核内,穿膜效率为40.86%,并且可与目的DNA进行特异结合,为蛋白体外诱导iPS的产生奠定基础。Objective： To prepare recombinant protein PTD -Sox2 with cell penetrating peptide and identify its activity. Method：The Sox2 gene was expressed as a fusion protein PTD - Sox2 with PTD using prokaryotic expression vector pKYB. The fusion protein was purified by Ni affinity chromatography and identified by Western blot analysis. The penetrating ability of the fusion protein PTD - Sox2 into CHO cells was investigated using fluorescein （ FITC ） labeling method. And the bioactivity of PTD - Sox2 to bind to the target DNA sequence was detected by fluorescence resonance energy transfer （FRET）. Result： The recombinant expression strain pKYB - PTD - Sox2 - 6His - ER2566 was successfully constructed, the fusion protein PTD-Sox2 was prepared and purified, its activity was identified. Conclusion： This research has proved that PTD - Sox2 fusion protein can not only penetrate the cell membrane and nuclear membrane, enter the nuclei with the efficiency of 40.86 ＋ 1.97%, but also combine with its target DNA sequence specifically. These works laid a good foundation for inducing of iPS cells with protein in vitro.

关 键 词：PTD—Sox2 iPS 核转录因子 原核表达 FITC FRET 

分 类 号：Q786[生物学—分子生物学]