一种人源性穿膜肽融合蛋白中连接肽的引入及其在HeLa细胞中的穿膜活性

Introduction of Linking Peptides to a Human-derived Cell-penetrating Peptide and Its Transduction Activity in HeLa

p14ARF(alternate reading frame)是一种人肿瘤抑制因子,其N端1-22位氨基酸可以携带药物蛋白进入细胞发挥药理作用,记为ARF。在示踪蛋白EGFP和ARF间引入了二肽(linker 2)、柔性七肽(linker 7F)和刚性七肽(linker 7S),研究其对融合蛋白EGFP-ARF穿膜活性的影响。实验结果显示引入七肽(linker 7)的融合蛋白,其荧光强度高于linker 2的融合蛋白。激光共聚焦观察表明,引入linker 7F的融合蛋白比引入linker 2的融合蛋白的穿膜效率高5倍左右,比引入linker 7S的融合蛋白高3.8倍左右。结果表明连接肽的形式对融合蛋白的结构和功能都有显著影响,为ARF作为药物运输载体的研究提供了依据。

p14ARF （alternate reading frame） is a human tumor inhibition factor .The first 22 amino acids, denoted by ARF ,can be absorbed by cells and then exert its function .In this study both of a 2 amino acids lin-ker peptide （ linker 2） ,a stable and flexible linking peptide of 7 amino acids （ linker 7S,linker 7F） were in-troduced to the site between Enhanced Green Fluorescence Peptide （EGFP）,a tracer peptide and ARF.The effect of three different linkers on recombinant protein EGFP-ARF was investigated .The results show that fluo-rescence intensity of proteins with 7 amino acids linker （ linker 7） is higher than that of linker 2,which means the length of linking peptide remarkably affects the function of EGFP .In virtue of laser confocal microscope ,the membrane-crossing activity is gained with the increase of linker length .Among this ,the uptake efficiency of re-combinant protein with linker 7F is 3.8 times higher than that of stable linking peptide and 5 times higher than that of linker 2.This study supplies a preferable construction method for using ARF as a valid carrier .