细胞穿透肽PEP-1介导增强型绿色荧光蛋白在小鼠体内跨膜转导

Cell-penetrating peptide PEP-1 mediated transmembrane delivery of enhanced green fluorescent protein in vivo of mouse

目的研究细胞穿透肽PEP-1介导的大分子物质在小鼠体内的跨膜转导能力。方法用基因工程的方法制备并纯化增强型绿色荧光蛋白EGFP和PEP-1-EGFP融合蛋白,分别将500μg的EGFP蛋白和PEP-1-EGFP融合蛋白通过尾静脉注射入昆明小鼠体内,2 h后麻醉小鼠,PBS充分灌流并取心、脑、肝、脾、肾快速冷冻切片后立即置荧光显微镜下观察。结果2 h后PEP-1-EGFP融合蛋白处理的小鼠大脑、心肌、肝、脾和肾组织里出现均一的明亮绿色荧光,而EGFP蛋白处理的小鼠各脏器内均未见到绿色荧光。结论细胞穿透肽PEP-1能携带增强型绿色荧光蛋白穿透小鼠细胞膜并分布于心、脑、肝、脾、肾组织内,为将来用PEP-1介导各种大分子药物跨膜转导进行各种疾病的蛋白治疗提供实验依据。

Objective To investigate the in vivo transduction capability of fusion protein PEP-1-EGFP with mice. Methods Two prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 （ 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. Five hundred micrograms of EGFP and PEP-1-EGFP fusion protein were injected into mouse through caudal vein, respectively, the mice were euthanized and perfused with PBS 2 hours after administration. Then, the heart, brain, liver, spleen and kidney were removed and sectioned with a cryostat at 7μm for visualization with a inverted fluorescent microscope. Results The brain, heart, liver, spleen and kidney injected with PEP-1-EGFP showed bright and homogenous green fluorescence whereas that with EGFP showed no green fluorescence at all. Conclusion The successful expression and purification of PEP-1-EGFP fusion protein and its efficient transduction into mice in vivo provide a basis for the research on transmembrane delivery of macromolecule drugs mediated by the cell-penetrating peptide, PEP-1.