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通过基因工程构建重组表达质粒pET15b-R9-FOXM1(1-234aa),转化大肠杆菌建立构建表达R9-FOXM1(1-234aa)的菌株.采用原核表达系统和His-tag亲和纯化手段,规模化制备纯化穿膜肽R9-FOXM1(1-234aa),获得的蛋白的纯度达到90%以上.用R9-FOXM1(1-234aa)穿膜肽处理不同的肿瘤细胞,通过MTT实验研究其细胞效应.结果显示:当R9-FOXM1(1-234aa)穿膜肽的浓度达到2mM时,肿瘤细胞的死亡率为50%左右.实验表明穿膜肽R9-FOXM1(1-234aa)抑制不同肿瘤细胞的生长,有可能成为治疗肿瘤的潜在蛋白类药物.  相似文献   
2.
Delivery of macromolecules into living cells by arginine-rich cell penetrating peptides (AR-CPPs) is an important new avenue for the development of novel therapeutic strategies. However, to date the mechanism of this delivery remains elusive. Recent data implicate endocytosis in the internalization of AR-CPPs and their macromolecular cargo and also indicate limited delivery of macromolecules into the cell cytoplasm and nucleus. Different types of endocytosis – clathrin-dependent endocytosis, raft/caveolin-dependent endocytosis and macropinocytosis – are all implicated in the uptake of AR-CPPs and their cargo into different cells. Cationic AR-CPPs dramatically increase uptake of conjugated molecules through efficient binding to surface proteoglycans. Whether this increase in binding can assure delivery of a sufficient amount of functionally active macromolecules into the cytoplasm and nucleus or whether there is a specific mechanism by which AR-CPPs facilitate the escape of conjugated cargo from endosomes remains to be understood. Received 30 June 2005; received after revision 9 August 2005; accepted 30 August 2005  相似文献   
3.
Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polye- lectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candi- date as a safe and efficient vector for gene delivery and transfection.  相似文献   
4.
Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and ^13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.  相似文献   
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