摘要
目的:构建原核表达载体pET15bPEP1SOD1,观察表达的融合蛋白PEP1SOD1能否进入细胞.方法:以pBluescriptIISKSOD1质粒为模板,PCR扩增SOD1cDNA.经酶切后分别构建原核表达载体pET15bSOD1和pET15bPEP1SOD1,分别转化大肠杆菌,表达SOD1和PEP1SOD1融合蛋白,将鉴定和纯化后的融合蛋白加入体外培养的人脐静脉内皮细胞.结果:得到高纯度的、有活性的SOD1和PEP1SOD1融合蛋白.PEP1SOD1融合蛋白能高效地转导入体外培养的人脐静脉内皮细胞,并表现出剂量依赖性和时间依赖性的特点;一旦进入细胞,PEP1SOD1融合蛋白可以维持48h.结论:PEP1SOD1融合蛋白能高效地转导入体外培养的人脐静脉内皮细胞,为SOD1进入组织细胞内发挥治疗作用提供了实验基础.
AIM： To construct a prokaryotic expression system of pET15b-PEP-1-SOD1, and to determine whether the expressed PEP-1-SOD1 fusion protein can be transduced into cells. METHODS： SOD1 cDNA was amplified by PCR using pBluescript Ⅱ SK-SOD1 as template, and digested with restriction endonuclease to construct recombinant plasmids pET15h-SOD1 and pET15b-PEP-1-SOD1, followed by being respectively transformed into E. coli to produce SOD1 and PEP-1-SOD1 fusion proteins, which were purified and added in cultured endothehal cells of human umbilical vein in vitro. RESULTS： The highly purified and active SOD1 and PEP-1-SOD1 fusion proteins were obtained. The PEP-1-SOD1 fusion protein could enter endothehal cells of human umbilical vein in a time- and dose-dependent manner when added exogenously in a culture media. Once inside the cells, PEP-1-SOD1 protein was enzymatically stable for 48 h. CONCLUSION： PEP-1-SOD1 fusion protein can be transduced effectively into endothelial cells of human umbilical vein, which provides a basis for future study involving direct delivery of SOD1 protein into tissues for therapy.
出处
《第四军医大学学报》
北大核心
2006年第9期855-859,共5页
Journal of the Fourth Military Medical University
关键词
超氧化物歧化酶
原核表
PEP-1肽
融合蛋白
superoxide dismutase
prokaryotic expression
PEP-1 peptide
fusion protein
