PEP-1-EGFP融合蛋白的表达和纯化及其转导入人脐静脉内皮细胞

目的 表达并纯化融合蛋白PEP—1-EGFP以对其穿透细胞膜的能力进行研究。方法 用分子克隆技术构建出表达型载体pET15b—pep—1—EGFP，在大肠杆菌Ecoli BL21（DE3）中表达融合蛋白PEP—1—EGFP并进行Ni^2＋-树脂柱亲和层析纯化以进行融合蛋白穿透人脐静脉内皮细胞的实验。结果 经测序证实成功构建了表达型载体pET15b—pep—1—EGFP,PEP—1—EGFP融合蛋白在大肠杆菌BL21（DE3）中得到高效表达，纯化后的蛋白浓度为6．18mg／ml，蛋白产量为14．15mg／100ml菌液，SDS—PAGE和Western blotting显示纯化蛋白为融合蛋白PEP—1—EGFP，细胞膜穿透实验证实PEP—1—EGFP融合蛋白能进入人脐静脉内皮细胞。结论 PEP—1—EGFP融合蛋白的表达和纯化及其穿膜能力的研究为PEP-1携带有生物活性的大分子物质进行蛋白治疗奠定了基础。

Expression and purification of PEP-1-EGFP fusion protein and its transduction into human umbilical vein endothelial cells

Objective To construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E.coli BL21(DE3) for evaluating the cell-penetrating capability of the cell-penetrating peptide PEP-1. Methods Two oligonucleotides encoding PEP-1 was synthesized and annealed to generate PEP-1-encoding DNA. The recombinant plasmid pET15b-pep-1-EGFP was constructed by inserting PEP-1-encoding DNA and enhanced green fluorescent protein (EGFP) cDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E.coli BL21(DE3) was purified with Ni2 -resin affinity chromatography and transduced into human umbilical vein endothelial cells. Results Sequence analysis confirmed successful construction of the expression vector pET15b-pep-1-EGFP, and the fusion protein PEP-1-EGFP was expressed and purified efficiently with a yield of approximately 14.15 mg/100 ml bacteria medium. SDS-PAGE and Western blotting identified the purified protein as PEP-1-EGFP, and the cell-penetration assay verified that the fusion protein could be transduced into human umbilical vein endothelial cells. Conclusion The successful expression and purification of PEP-1-EGFP and its efficient transduction into human umbilical vein endothelial cells provides a basis for PEP-1-mediated biomacromolecular transduction in protein therapy.