基于宏基因组的新型启动子诱捕系统构建及在Nissle1917表达系统改进中的应用

项目名称： 基于宏基因组的新型启动子诱捕系统构建及在Nissle1917表达系统改进中的应用

项目编号： No.31300090

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： 杨继建

作者单位： 泰山学院

项目金额： 17万元

中文摘要： 目前能够商品化应用的高效表达启动子系统较少，仅限于少数的宿主且诱导成本高。本项目是基于环境宏基因组中含有大量生物资源的理论研究基础，结合申请者前期研究成果，以有机磷水解酶基因（mpd）和融合基因（mpd-gfp）为双向报告基因构建一新颖的启动子诱捕系统。该启动子诱捕系统具有灵敏、双向和简单等优点，适用于环境宏基因中启动子大通量筛选。针对益生菌Nissle1917表达系统，本项目建立环境宏基因随机启动子文库，通过分析启动子功能片段，建立一套Nissle1917高效表达和分泌系统。其成果可解决目前Nissle1917重组表达中常见的表达水平低、诱导剂成本高、包涵体形成等问题，为利用细菌Nissle1917在体内特异性传递外源药物提供一个良好的平台，为细菌靶向治疗肿瘤打下基础。

中文关键词： 厌氧启动子；Nissle1917；大肠杆菌；启动子探针；宏基因组

英文摘要： Currently, a few promoter systems are commercially available and effective in a limited range of hosts. However, they also require an expensive inducer. In this study, A novel promoter trapping vector will be constructed with the promoterless methyl parathion hydrolase gene (mpd) and mpd-gfp as the bidirectional reporters. The system has advantages of sensitivity, bidirection and simplicity so that it can be used easily to clone promoters with promoter strength and fluoresce on selective plates. As for probiotic Nissle1917，research is urgently needed to identify new promoters and develop novel expression systems. In this study,a metagenome random promoter library is constructed with this vector. Promoter regions with strong promoting and signal peptide functions are cloned from metagenomic promoter library. Based on the above observations, some constitutive promoters will be employed in the expression vector system and their potentials for practical application are evaluated in terms of expression level, protein solubility, and effects on host growth. This study will give advantages in high expression levels, low costs for expression induction, and reducing the formation of inclusion bodies. This new Nissle1917 expression systems will provide advanced approach for us to deliver exogenous medicine and treat tumor

英文关键词： anaerobic promoter；Nissle1917；E.coli；promoter probe；metagenome