PEP-1介导增强型绿色荧光蛋白转导入人主动脉平滑肌细胞

Cell-penetrating Peptide PEP-1-mediated Transduction of Enhanced Green Fluorescent Protein into Human Aortic Smooth Muscle Cells

目的:研究细胞穿透肽PEP-1携带大分子蛋白穿透人平滑肌细胞的能力。方法:用基因工程的方法制备并纯化EGFP蛋白和PEP-1-EGFP融合蛋白,将纯化后的两种蛋白和培养的人平滑肌细胞共同孵育,在荧光显微镜下直接观察PEP-1介导目的蛋白EGFP转导入人平滑肌细胞的能力。结果:EGFP蛋白不能进入细胞内,PEP-1-EGFP融合蛋白和人平滑肌细胞共同孵育1h后可见有明亮绿色荧光分布于胞浆和胞核。结论:PEP-1能有效携带目的蛋白进入人平滑肌细胞,这为将来用细胞穿透肽PEP-1介导有生物活性的大分子抗平滑肌细胞增殖进行血管成形术后再狭窄的蛋白治疗奠定了基础。

Objective To investigate the penetrating ability of fusion protein PEP- 1 -EGFP with human aortic smooth muscle cells. Methods Two prokaryotic expression plasmids pET15b - EGFP and pET15b - PEP - 1 - EGFP were constructed and transformed into E. coli BI21 （ DE3 ） to express EGFP and fusion protein PEP - 1 - EGFP, respectively. The expressed EGFP and PEP - 1 - EGFP were purified with Ni^ 2＋ resin affinity chromatography and their capability of transduction into human aortic smooth muscle cells was evaluated with inverted fluorescent microscope. Results EGFP alone failed to trans- duce into human aortic smooth muscle cells, whereas PEP - 1 - EGFP fusion protein could transduce into them and distributed in cytoplasm and nucleus after lh incubation. Conclusion The successful expression and purification of PEP - 1 - EG- FP fusion protein and its efficient transduction into human vascular smooth muscle cells provides a basis for the research on transduction of antiproliferative proteins such as p27 and p21 mediated by the cell - penetrating peptide, PEP - 1, in protein therapy for the restenosis after angioplasty.