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Novel gene delivery systems using synthetic oligopeptide and MAP

使用合成寡肽和 MAP 的新型基因传递系统

基本信息

批准号：
09672195
负责人：
HAZEMOTO Norio
金额：
$ 1.98万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672195/
关键词：
synthetic peptide MAP DNA gene transfer transfection amino acid composition オリゴペプチドプラスミドDNA

项目摘要

We describe a novel approach for gene transfer mediated by synthetic peptides. This method was base on use of dendritic peptides that developed as the multiple antigen peptides(MAP). The tetra and octameric branched peptide as transfection reagent were compared with the monomeric peptide consisted of lysine residues, polylysine. The monomeric peptide was essentially little activity of transfection, near the background level, but in the dendritic peptides, especially, octameric peptides showed higher transfection efficiency. Effects of chain length of branched lysine residue were studied using 3, 6, 9, 12, 15 mer octameric peptides. Short length peptides up to 6 mer were not efficient, but 12 or 15 mer peptides were more efficient for DNA transfection. The ability of the dendritic peptides on DNA transfection was examined in six cultured mammalian cells : HeLaS3, NIH-3T3, L929, CV-l, COS-7 and HepG2. The peptides yielded an efficient gene transfer with a variety of cell lines. These stu … More dies suggested that branched structure in the cationic peptide was more favorable for efficient gene delivery.Oligopeptide of various sequences constituted by 9 amino acids residue were synthesized by F-moc chemistry. They contain lysine, leucine, tryptophan, cysteine or serine. The transfection ability of these synthetic oligo-peptides were examined in terms of the functional transfer of pSV2cat plasmid DNA into cells. Specific oligopeptide composed of lysine, tryptophan and cystein have ability (facilitate) of gene transfer to mammalian cells. Dimer of KLKLCLKLK substituted a part of lysine by leucine showed somewhat expression. A substitution a part or all of leusine to tryptophan still more promote gene delivery, especially dimer of KWKWCWKWK showed highest efficient transfection. These results indicated hydrophobic amino acid as well as cationic amino acid is necessary for gene delivery by oligopeptide and tryptophan is more favorable than leucine. Furthermore, it is worthy of notice that CWKWKWKWK and KWKWKWKWC which possess N or C-terminal cysteine residue and form linear dimer not at all mediate gene delivery. It indicates that the structure of dimer peptide produced by formation of S-S bond is critical in deciding whether to deliver DNA to cells. It suggests that Trp pairs in KWKWCWKWK dimer is in particular close each other and provides the stronger couplet. The CD spectra of KWKWCWKWK dimer reflected their distinctive structure. Less

我们描述了一种由合成肽介导的基因转移的新方法。该方法是利用树突状多肽作为多抗原肽(MAP)的基础上发展起来的。将四聚体和八聚体支链多肽作为转染剂，与由赖氨酸残基、多聚赖氨酸组成的单体多肽进行比较。单体多肽的转染率基本不高，接近本底水平，但在树突状多肽中，尤其是八聚多肽显示出较高的转染率。研究了3，6，9，12，15聚体八聚体对支链赖氨酸残基链长的影响。6聚体以下的短肽效率较低，12或15聚体的DNA转染率较高。在HeLaS_3、NIH-3T_3、L929、CV-L、COS-7和HepG_2等6种哺乳动物细胞中检测了树突状多肽的DNA转导能力。这些多肽与多种细胞系进行了有效的基因转移。这些STU…更多的数据表明，阳离子多肽中的分支结构更有利于基因的高效传递。利用F-moc化学合成了由9个氨基酸残基组成的各种序列的寡肽。它们含有赖氨酸、亮氨酸、色氨酸、半胱氨酸或丝氨酸。通过将pSV2cat质粒DNA功能转移到细胞内来检测这些合成的寡肽的转染性。由赖氨酸、色氨酸和半胱氨酸组成的特异性寡肽具有向哺乳动物细胞转移基因的能力。用亮氨酸取代部分赖氨酸的KLKLCLKLK二聚体有一定的表达。将部分或全部亮氨酸替换为色氨酸仍能更好地促进基因的传递，尤其是KWKWCWKWK的二聚体具有最高的转染率。这些结果表明，疏水氨基酸和阳离子氨基酸是寡肽基因传递所必需的，色氨酸比亮氨酸更有利于基因的传递。此外，值得注意的是，具有N或C端半胱氨酸残基并形成线性二聚体的CWKWKWWWK和KWKWKWWWC根本不介导基因的传递。这表明，S-S键的形成所产生的二聚肽的结构是决定是否将DNA输送到细胞中的关键。这表明KWKWCWKWK二聚体中的色氨酸对尤其紧密，并提供了更强的偶联。KWKWCWKWK二聚体的CD光谱反映了它们独特的结构。较少