VP22 MEDIATED DELIVERY OF PROTEIN AND DNA INTO CELLS

这项提案概述了VP22作为一种将蛋白质和DNA分子运送到培养细胞的载体的发展。第一阶段的提案通过表达和纯化与来自大肠杆菌的触角线虫多肽的融合蛋白，展示了蛋白质介导的传递的力量。这些融合蛋白通过生物膜转运，积聚在细胞质和细胞核中。最近发现了一种新的转位蛋白VP22，它具有两个显著的优点：1)VP22可以转位较大的多肽；2)VP22在细胞亚群中的表达导致该蛋白在整个培养过程中扩散。在第二阶段，将构建VP22融合蛋白的原核和真核表达载体。VP22的融合将被用于开发能够调节基因表达的系统。与DNA相互作用的VP22融合将被开发并用于研究VP22向培养细胞运送寡核苷酸和质粒DNA的能力。这项革命性的技术在将蛋白质和DNA输送到细胞方面有着广泛的应用，这是目前的技术无法转化的，并将为研究所有细胞类型的基因功能提供新的技术。拟议的商业应用：将构建原核和真核载体，以允许研究人员表达和纯化VP22融合蛋白。将构建VP22融合，以开发可诱导的基因表达系统，并将单链抗体输送到细胞内靶点。基于VP22的试剂将被开发用于将寡核苷酸和质粒DNA输送到组织培养细胞，这些细胞对当前的转基因技术是困难的。

This proposal outlines the development of VP22 as a delivery vehicle for protein and DNA molecules to cells in culture. The Phase I proposal demonstrated the power of protein-mediated delivery by expressing and purifying protein fusions with the Antennapedia peptide from E. coli. These fusion proteins were translocated across biological membranes, accumulating in the cytoplasm and nucleus. A new translocating protein, VP22, was recently described and has two significant advantages over the Antennapedia peptide: 1) VP22 can translocate larger peptides and 2) expression of VP22 in a subpopulation of cells results in spread of the protein throughout the entire culture. During the Phase II period, prokaryotic and eukaryotic vectors for the expression and purification of VP22 fusion proteins will be constructed. VP22 fusions will be used to develop systems that can regulate gene expression. VP22 fusions that interact with DNA will be developed and used to investigate the ability of VP22 to deliver oligonucleotides and plasmid DNA to cells in culture. This revolutionary technology has broad applications for the delivery of proteins and DNA to cells that cannot be transfected with current technologies and will provide new techniques to study gene function in all cell types. PROPOSED COMMERCIAL APPLICATIONS: Prokaryotic and eukaryotic vectors will be constructed to allow researchers to express and purify VP22 fusion proteins. VP22 fusions will be constructed to develop an inducible gene expression system and to deliver single chain antibodies to intracellular targets. Reagents based on VP22 will be developed for the delivery of oligonucleotides and plasmid DNA to tissue culture cells that are refractory to current transfection technologies.