由胞外向内质网定向递送蛋白的细胞生物学工具的研发和应用的探讨

本项目拟以霍乱外毒素和志贺外毒素的细胞内转运的机理为理论基础，利用这类细菌外毒素的序列片段设计能够诱导重组蛋白从细胞外向内质网网腔转运的蛋白标签；然后优化蛋白标签的序列，并通过引入内质网内源性信号肽酶的酶切位点来改造蛋白标签，降低运输载体对于所递送蛋白的干扰。以此为基础，本项目将进一步对这一蛋白标签的应用进行探究并构造一系列功能性的带有内质网转运标签的重组蛋白。. 这一细胞生物学工具的研发可以为阐明与内质网密切相关的一系列生物化学过程和信号传导的机制提供帮助，并为开发与内质网网腔驻留蛋白相关的遗传病的蛋白药物奠定基础。

This project aims to establish a protein delivery tool for delivering protein of interest from extracellular space into the lumen of endoplasmic reticulum (ER) of mammalian cells. We propose to develop a protein tag based on cholera toxin or shiga toxin and hijack the retrograde transport mechanism of the toxins. We will optimize the tag sequence and introduce an ER signal peptide cleavage site for releasing of the target protein after reaching ER. Upon development of the protein tag, we also intend to make use of the protein tag and investigate the functional consequence after delivering a series of tag fused ER luminal proteins into cells. . The successful establishment of this method can help elucidate ER related cell signaling pathways and biochemistry process in ER lumen. It will also facilitate the development of protein drugs towards treatment of genetic disorders involving ER luminal proteins.