INTERNALIZATION OF PEPTIDE FUSIONS BY MAMMALIAN CELLS

这项建议概述了一个系统的发展，引入肽 转化为哺乳动物组织培养细胞。这 系统利用源自果蝇的16个氨基酸的肽 一种跨神经元膜易位的足同源蛋白 当在细胞外孵育时。这种肽的内化并不 似乎是由内吞作用介导的或需要受体，这表明 它可以用于多种细胞类型。原核表达 构建载体，用于成本有效的合成和纯化 融合肽。肽融合的易位 将测试不同的尺寸以确定这种结构的空间限制。 系统这些肽的易位也将用许多 常见的组织培养细胞系，以证明这一技术的实用性。 与需要将肽直接注射到细胞中的技术不同， 将该肽载体导入细胞是快速和有效的。 一旦内化，这种肽在细胞质和细胞外基质中被发现。 原子核该技术具有广泛的应用，包括 抑制性或治疗性肽的递送， 寡核苷酸和基因治疗。 拟定商业应用： 第一阶段的研究将导致原核质粒载体， 简单的表达和纯化的双足肽融合。这些 肽可用于将肽或寡核苷酸引入细胞 快速有效地新闻媒体技术可以应用于 许多学科的研究，包括基因转移和基因治疗。

This proposal outlines the development of a system to introduce peptides into mammalian tissue culture cells in a rapid and efficient manner. This system utilize a 16 amino acid peptide, derived from the Drosophilia Antennapedia homeoprotein, that is translocated across neuronal membranes when incubated extracellularly. Internalization of this peptide does not appear to be mediated by endocytosis or require a receptor, suggesting that it may be useful in a variety of cell types. A prokaryotic expression vector win he constructed for cost-efficient synthesis and purification of Antennapedia fusion peptides. Translocation of peptide fusions of different sizes will be tested to determine the steric constraints of this system. The translocation of these peptides will also be tested with many common tissue culture cell lines to prove the utility of this technology. Unlike techniques requiring direct injection of peptides into cells, introduction of this peptide vector into cells is rapid and efficient. Once internalized, this peptide is found in both the cytoplasm and nucleus. This technology has a wide variety of applications, including the delivery of inhibitory or therapeutic peptides, the delivery of oligonucleotides, and gene therapy. PROPOSED COMMERCIAL APPLICATION: Phase I research will result in a prokaryotic plasmid vector that allows simple expression and purification of Antennapedia peptide fusions. These peptides can be used to introduce peptides or oligonucleotides into cells quickly and efficiently. The Antennapedia technology can he applied to research in many disciplines, including gene transfer and gene therapy.