HrpN基因在毕赤酵母中的克隆表达与活性检测  

Expression and Biological Assay of HrpN in Pichia pastoris

作　　者：黄世容[1] 孟小林[1] 徐进平[1] 鲁伟[1] 王健[1] 

机构地区：[1]武汉大学生命科学学院病毒研究所,武汉430072

出　　处：《武汉植物学研究》2005年第4期376-380,共5页Journal of Wuhan Botanical Research

基　　金：香港绿色生命实验室资助项目。

摘　　要：将hrpN基因插入毕赤酵母表达载体pPIC6αA的醇氧化酶(AOX1)启动子下游,构建重组表达载体pPIC6αA-hrpN,经电击转化毕赤酵母菌株X-33和杀稻瘟素筛选,得到高效分泌表达harpinEa的毕赤酵母菌株。经初步纯化、SDS-PAGE分析和生物测定,结果显示,诱导96h的此重组菌大量表达harpinEa,表达产物具有诱导烟草超敏感反应的功能,活性高于在大肠杆菌中表达的harpinEa。HrpN gene was placed under the control of AOX1 promoter in a Pichia expression vector pPIC6αA. Through electroporation transformation of X-33 Pichia and Blasticidin selection, a strain of Pichia pastoris capable of secreting harpin Ea into the medium at high level was obtained. After initially purified, SDS-PAGE analysis of the expression products indicated that harpinEa was secreted at high level when the Pichia pastoris expression clone had been induced for 96 hours. The results of bioassay showed that the recombinant harpin Ea had the ability of eliciting hypersensitive response in tobacco. The activity of this harpinEa is higher than that of harpinEa expressed in Escherichia coli.

关 键 词：毕赤酵母 分泌表达 融合蛋白 活性检测 

分 类 号：Q78[生物学—分子生物学]