融合表达载体pET32a/Trx-EK-MrVIB的构建及在大肠杆菌中表达  

The Construction of Fusion Expression Vector p ET32a/Trx-EK-Mr VIB and Expression in Escherichia coli

作　　者：高炳淼[1] 刘云海[1] 彭超 魏娜[1] 张俊清[1] 

机构地区：[1]海南医学院药学院,海南省热带药用植物研究开发重点实验室,海口571199 [2]深圳华大基因研究院,深圳518083

出　　处：《基因组学与应用生物学》2015年第2期326-331,共6页Genomics and Applied Biology

基　　金：海南医学院培育基金(HY2013-11);海南医学院大学生创新课题(HYCX201327);海南省自然科学基金(814291);海南医学院引进人才科研启动经费;国家高技术研究发展计划(863计划)课题(2014AA093501)共同资助

摘　　要：人工设计合成芋螺毒素基因Mr VIB来构建表达载体p ET32a/Trx-EK-Mr VIB,将其转化大肠杆菌BL21(DE3)plys S进行诱导表达。菌体经超声破碎后利用Ni-NTA琼脂糖柱进行亲和层析纯化融合蛋白,SDS-PAGE电泳分析融合蛋白表达。结果表明融合表达载体p ET32a/Trx-EK-Mr VIB经PCR扩增和测序鉴定具有正确的开放阅读框。SDS-PAGE电泳显示融合蛋白在大肠杆菌中获得高效可溶性表达,经一步亲和层析获得纯度大于90%的融合芋螺毒素达73.6 mg/L。本文成功构建了融合表达载体p ET32a/Trx-EK-Mr VIB,融合芋螺毒素Trx-EK-Mr VIB在大肠杆菌中获得高效可溶性表达。Artificial designed conotoxin MrVIB gene was synthesized to construct expression vector p ET32a/TrxEK-MrVIB, which was transformed into E. coli BL21（DE3） pLysS and induced expression by IPTG. Recombinant fusion proteins were purified by affinity chromatography using Ni-NTA agarose column, the expression and purification of fusion proteins to be analyzed by SDS-PAGE. Fusion expression vector pET32a/Trx-EK-MrVIB was confirmed correctly by PCR and sequence analysis. The result of SDS-PAGE showed that fusion protein was high effectively expressed in E. coli, which was purified by one-step affinity chromatography and the purity of fusion conotoxin was greater than 90% and its yield was 73.6 mg/L. Fusion expression vector pET32a/Trx-EK-MrVIB was successfully constructed, and fusion conotoxin Trx-EK-MrVIB was high effectively expressed in E. coli.

关 键 词：芋螺毒素 大肠杆菌 基因工程 融合表达 亲和层析 

分 类 号：Q78[生物学—分子生物学]