微环-附着体表达载体系统的适配性及其机制

项目名称： 微环-附着体表达载体系统的适配性及其机制

项目编号： No.31300702

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 王小引

作者单位： 新乡医学院

项目金额： 22万元

中文摘要： 表达载体在基因治疗中起着关键的作用，基因治疗目前采用的载体都存在一定的缺陷。微环-附着体表达载体（mincircle episiome vector）是申请者前期构建的一种新型的哺乳动物细胞表达载体，可以消除原核元件、附着而非整合到中国仓鼠卵巢细胞(CHO)染色体上，克服传统载体的原核元件及整合效应带来的副作用，安全、高效、稳定地驱动转基因表达，已获得国家授权发明专利。载体的适配性及其机制尚待进一步研究。本项目拟对微环-附着体表达载体的关键调控元件、宿主细胞的适配性及其机制进行研究。拟采用分子生物学技术分别克隆不同的启动子，分析载体与不同启动子的适配性，探讨载体适配的通用高效的启动子；进而探讨载体与宿主细胞的适配性。分析转基因拷贝数和整合位点、应力诱导的双重不稳定性及细胞转化株的染色质构象变化，为建立新型的基因治疗载体提供理论和实验基础。

中文关键词： 附着体载体；基因表达；启动子；宿主细胞；适配性

英文摘要： Expression vector play a key role in gene therapy, but the current vectors used have some limitaion. In our previous studies, we established one new kind of expression vector: minicircle episomal vector, which could eliminate the bacterial elements and episomal exist, not integrate into the chromosome of Chinese hamster ovary (CHO) cells. The vector could overcome the side effect of the traditional expression vector,which caused by the bacterial elements and integration effect. The patent of this vector has been authorized. However, the compatibility and mechanism of the vector has been unclear. This project will study the compatibility and mechanism of the key regulator elements and host cell expression system. The present study will use the molecular technology to clone different promoters and study the compatibility between promoters and vector,to explore the universal effective promoter. Different target cells will be utilizied to explore their compatibility. Transgenic copy numbers and integration sites will further be analyzed. Finally, we will focus on the stress-induced duplex destabilisation and chromatin conformation of stably transgenic cells mediated by MAR. This study will provide theoretical and experimental basis for establishing the new gene therapy vector.

英文关键词： episomal vector；gene expression；promoter；host cell；compatibility