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

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.