小鼠Nanog基因原核表达载体的构建及表达.pdfVIP

中国农业科学 2007,40(2):373-378 Scientia Agricultura Sinica 小鼠Nanog基因原核表达载体的构建及表达 李 军，吕长荣，窦 琳，窦忠英 （西北农林科技大学国家干细胞工程技术研究中心陕西分中心，杨凌 712100 ） 摘要：【目的】构建小鼠Nanog基因原核表达载体并进行表达，以期得到大量GST融合蛋白。【方法】根据GeneBank 中的Nanog序列及pGEX-KG中的多克隆位点设计引物，以含有Nanog基因片段的pNA992重组质粒为模板，经PCR扩增 出918 bp的DNA片段。将所得片段与pGEX-KG载体连接，转化TGⅠ大肠杆菌，筛选阳性克隆，其扩增片段测序结果 与原序列一致，表明原核表达载体pGEX-KG-Nanog已构建成功。提取pGEX-KG-Nanog质粒转化到BL21（DE3）表达菌 株中，经IPTG诱导后收集菌体进行SDS电泳鉴定，并优化其表达条件。【结果】在大肠杆菌中获得Nanog基因 融合表达，主要以包涵体形式存在；融合蛋白的分子量为 63 kD；以IPTG终浓度为 0.8 mmol·L-1 ，诱导 5 h后融合 蛋白产量最高。【结论】小鼠Nanog基因在大肠杆菌中获得了高效表达，为今后Nanog蛋白的多克隆抗体制备奠定 基础。 关键词：Nanog基因；原核表达；GST融合蛋白；小鼠 Construction of Prokaryotic Expression Vector of Mouse Nanog Gene and Its Expression LI Jun ，LÜ Chang-rong ，DOU Lin ，DOU Zhong-ying (Northwest Sci-Tech University of Agriculture and Forestry, Shaanxi Branch of National Stem Cell Engineering and Technology Center, Yangling 712100) Abstract: 【Objective 】Prokaryotic expression vector of mouse Nanog gene was constructed and was expressed to obtain GST fusion protein. 【Method 】A pair of primers were designed according to digestion sites in plasmid pGEX-KG and the Nanog gene sequence published by GeneBank. The DNA fragment of 918 bp was amplified by PCR from the pNA992 recombinant plasmid with Nanog gene, then cloned into pGEX-KG and transformed into the host E .coli strain TG Ⅰ, The fragment was conformed to the original sequence. It indicated that fusion expression vector pGEX-KG-Nanog was constructed. The pGEX-KG-Nanog plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 ℃, the expression product of Nanog gene was identified by SDS, and the expression cond