INTERNALIZATION OF PEPTIDE FUSIONS BY MAMMALIAN CELLS

这项提案概述了一种引入多肽的系统的发展 以一种快速有效的方式转化为哺乳动物组织培养细胞。这 该系统利用从果蝇中提取的16个氨基酸的多肽 触角线虫同源蛋白，跨神经元膜转运 当细胞外孵化时。这种多肽的内化不会 似乎是由内吞作用介导的，或者需要受体，提示 它可能在各种类型的细胞中都有用。一种原核表达 为经济高效地合成和纯化而构建的载体Win 天牛融合多肽。多肽融合蛋白的易位 将测试不同的大小以确定这一点的空间约束 系统。这些多肽的移位也将在许多 普通组织培养细胞系，以证明这项技术的实用性。 与需要将多肽直接注入细胞的技术不同， 将这种多肽载体导入细胞是快速而有效的。 一旦内化，这种多肽在细胞质和 原子核。这项技术有广泛的应用，包括 抑制或治疗多肽的递送，递送 寡核苷酸和基因疗法。 建议的商业应用： 第一阶段的研究将产生一种原核质粒载体，使 触角虫肽融合蛋白的简易表达和纯化。这些 多肽可以用来将多肽或寡核苷酸引入细胞 又快又高效。天线技术可以应用于 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.