摘要
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E.coliBL21(DE3)中表达融合蛋白PEP-1-EGFP并进行Ni2+-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Westernblotting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。
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） eDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E.coli BL21 （DE3） was purified with Ni^2＋-resin affinity chromatography and transduced into human umbilical vein endothelial cells. Results Sequence analysis confirmed successful construction of the expression vector pET 15b-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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2006年第8期1114-1117,共4页
Journal of Southern Medical University
关键词
细胞穿透肽
增强型绿色荧光蛋白
原核表达
蛋白转导
人脐静脉内皮细胞
cell-penetrating peptides
enhanced green fluorescent protein
prokaryotic expression
protein transduction
human umbilical vein endothelial cell
