一种筛选原核高效表达克隆的方法  

Research of a screening strategy for high prokaryotic expression clone

作　　者：李福胜[1] 李玉富[1] 邵华[1] 张智清[1] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室

出　　处：《中华实验和临床病毒学杂志》1997年第4期311-314,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：外源基因在大肠杆菌中的低表达往往与基因５’端核苷酸序列不利于翻译起始有关。未经修饰的ＧＭ－ＣＳＦ在大肠杆菌中表达量很低，我们选用ＧＭ－ＣＳＦ作为报道基因，在不改变氨基酸序列的前提下，通过聚合酶链反应（ＰＣＲ）扩增，构建了两类包含５’端１０个密码子的简并序列库，为便于快速筛选高表达的克隆，构建了四环素抗性（ＴｅｔＲ）选择载体筛选系统，其基本原理是外源基因与ＴｅｔＲ基因串联，高表达的ＧＭ－ＣＳＦ５’端带动ＴｅｔＲ的表达，逐渐提高四环素浓度就可以筛选出适合高表达的ＧＭ－ＣＳＦ５’端序列。最终在四环素浓度高达６０μｇ／ｍｌ的培养基上筛选了数个高表达克隆。这种系统对大肠杆菌中表达量低。The low expression in E.coli can be often explained by the beginning sequence that causes low translational initiation. A simple selection procedure is then used to enrich those sequences from the bank that lead to high levels of translation. The selection procedure is based on the use of a translationally coupled tetracycline resistance gene. The basic steps are as follows: (1) Construction of selection vector pBV223; (2) Screening high expression clones through different tetracycline concentrations. Several clones have been selected in 60μg/ml concentration of tetracycline. So this system will provide an important way in preparation of some cytokines which have a low expression level in E.coli but have significant economic value.

关 键 词：原核表达 GM-CSF 四环素抗性基因 质粒 

分 类 号：R446.5[医药卫生—诊断学]