医学论文-PEP.docVIP

医学论文-PEP ????????????? 作者：丁鹏，王家宁，黄永章，骆丽娜，邵芳 【关键词】? ,超氧化物歧化酶 ??? Transduction of human Cu, Znsuperoxide dismutase mediated by an PEP1 peptide into endothelial cells of human umbilical vein 　　【Abstract】 AIM: To construct a prokaryotic expression system of pET15bPEP1SOD1, and to determine whether the expressed PEP1SOD1 fusion protein can be transduced into cells. METHODS: SOD1 cDNA was amplified by PCR using pBluescript II SKSOD1 as template, and digested with restriction endonuclease to construct recombinant plasmids pET15bSOD1 and pET15bPEP1SOD1, followed by being respectively transformed into E.coli to produce SOD1 and PEP1SOD1 fusion proteins, which were purified and added in cultured endothelial cells of human umbilical vein in vitro. RESULTS: The highly purified and active SOD1 and PEP1SOD1 fusion proteins were obtained.The PEP1SOD1 fusion protein could enter endothelial cells of human umbilical vein in a time and dosedependent manner when added exogenously in a culture media. Once inside the cells, PEP1SOD1 protein was enzymatically stable for 48 h. CONCLUSION: PEP1SOD1 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. 　　【Keywords】 superoxide dismutase； prokaryotic expression； PEP1 peptide；? fusion protein 　　【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pBluescript II SKSOD1质粒为模板, PCR扩增SOD1 cDNA. 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48 h. 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. 　　【关键词】 超氧化物歧化酶；原核表达；PEP1肽；融合蛋白 　　0引言 　　超氧化物歧化酶（superoxide dismutase, SOD）是清除细胞内自由基, 保护细胞免受氧化应激损伤的关键酶之一［1］. 哺乳类动物细胞含有3种SOD［2］，即胞浆中的Cu,ZnSOD（SOD1），线粒体中的MnSOD（SOD2）和细胞外的Cu,ZnSOD（ECSOD1, SOD3）. SOD1是细胞中高水平表达的SOD, 是SOD的主要形式. 长期以来人们希望应用SOD1来防