NF-κB相互作用多肽原核表达载体的构建、表达与纯化  

Construction,Expression,Purification of Prokaryotic Vector of Polypeptide Interacting with NF-κB

作　　者：史海水[1] 梁华平[2] 徐祥[2] 李生茂[2] 

机构地区：[1]河北医科大学生物化学教研室,河北石家庄050017 [2]第三军医大学大坪医院野战外科研究所第一研究室,重庆400042

出　　处：《河北大学学报（自然科学版）》2005年第5期530-534,共5页Journal of Hebei University(Natural Science Edition)

基　　金：国家重点基础研究发展规划项目(G1999054203);国家自然科学基金资助项目(30080000930200270);全军"十五"课题基金项目(2001-2005)

摘　　要：为了获得高纯度的可溶性NF_κB相互作用多肽,首先以酵母双杂交技术筛选获得的NF_κB相互作用多肽的酵母表达质粒pGAD GH/pp10为模板,扩增多肽基因片段,后经BglⅡ,StuⅠ双酶切后连接到pET_42a(+)载体中,构建GST/多肽融合蛋白的原核表达载体,并将GST/多肽融合蛋白的原核表达载体转化入BL_21菌株,用0.4 mmol/L IPTG于30℃诱导表达4 h,后经GST亲和纯化GST/多肽融合蛋白,SDS_PAGE电泳鉴定融合蛋白的表达和纯度.实验结果表明,成功地构建了NF_κB相互作用多肽的GST/多肽融合蛋白原核表达载体,进行了GST/多肽融合蛋白的诱导表达,融合蛋白的表达为可溶性表达,最终纯化获得纯度约为80%的可溶性GST/多肽融合蛋白,这将为后继利用GST pull_down,Bio_sensor,EMSA等实验进一步验证NF_κB相互作用多肽的功能提供可靠的材料来源奠定基础.To get purified soluble polypeptide interacting with NF-κB, Code fragment of polypeptide was obtained via PCR from pGAD GH/pp10 plasmid, which was screened by yeast two hybrid system previously. The fragment were inserted into pET-42a（ ＋ ） vector after digestion by restriction end-nuclease Bgl Ⅱ and Stu Ⅰ . Recombinant plasmid was transformed into BL-21 strain and induced 4 hours with 0.4 mmol/L IPTG at 30 ℃. The fusion protein was purified by GST affinity column and identif-ied by SDS-PAGE electrophoresis. Results showed that recombinant plasmid of GST-polypeptide fusion-protein was constructed successfully, after being induced and purified. Its final purity is about 80 %.

关 键 词：NF—κB 多肽 蛋白表达 

分 类 号：Q503[生物学—生物化学] Q51