带有穿膜肽PTD的重组Oct4的制备及活性鉴定  

Preparation and activity evaluation of recombinant Oct4 with penetrating peptide PTD

作　　者：李红阳[1,2,3] 李晓霞[1,2] 余榕捷[4] 刘晓飞[4] 王静静[4] 苏航[3] 陈建苏[1,2,3] 

机构地区：[1]暨南大学第一临床医学院,广东广州510632 [2]暨南大学医学院眼科,广东广州510632 [3]暨南大学再生医学教育部重点实验室,广东广州510632 [4]暨南大学生物工程研究所,广东广州510632

出　　处：《中国病理生理杂志》2011年第9期1790-1795,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30973244);中央高校基本科研业务费专项资金资助项目(No.21609407)

摘　　要：目的:重组蛋白构建细胞转录因子PTD-Oct4。方法:利用重叠延伸PCR法重组目的基因构建质粒pKYB-PTD-Oct4,并转染至E.coli ER2566。镍柱纯化重组蛋白并用Western bloting对其进行鉴定。用异硫氰酸荧光素(FITC)标记PTD-Oct4,检测其穿透中国仓鼠卵巢(CHO)细胞膜的能力;用荧光共振能量转移(FRET)检测重组融合蛋白PTD-Oct4结合目的DNA的活性。结果:成功获得目的蛋白PTD-Oct4,其可以成功进入细胞,定位于细胞核内,穿膜效率为(28.3±2.4)%,并具有与目标DNA序列特异结合的能力。结论:制备重组PTD-Oct4为外源蛋白诱导多能干细胞(IPSCs)奠定基础。AIM： To prepare recombinant protein of octamer-binding transcription factor-4（Oct4） with penetrating membrane activity.METHODS： Using overlap-extension PCR and prokaryotic expression vector pKYB,the Oct4 gene was expressed as a fusion protein combined with protein transduction domain（PTD） by gene splicing.The plasmid was transfected into E.coli ER2566.The fusion protein PTD-Oct4 was purified by Ni2＋ affinity chromatography and identified by Western blotting.The fluorescein isothiocyanate（FITC）-labeled PTD-Oct4 was used to investigate the penetrating ability of the fusion protein into Chinese hamster ovary（CHO） cells.The bioactivity of PTD-Oct4 binding to the target DNA sequence was detected by the method of fluorescence resonance energy transfer（FRET）.RESULTS： The recombinant PTD-Oct4 successfully entered the cells and located in the nucleus with the transmembrane efficiency of（28.3±2.4）%.The recombinant PTD-Oct4 had specific binding capacity with its specific target DNA sequence.CONCLUSION： The recombinant PTD-Oct4 was successfully prepared,which can be used to induce target cells to become induced pluripotent stem cells.

关 键 词：OCT4 诱导全能干细胞 

分 类 号：Q78[生物学—分子生物学]