pPIC9K-IL-2重组质粒的构建及其在毕赤酵母中的表达  

Construction of recombinant pPIC9K-IL-2 and expression in Pichia pastoris

作　　者：刘斌 祁光宇 陈晓宇 王宇 牟克斌[1,2] 黄银君[1,2] 刘学荣[1,2] 

机构地区：[1]中农威特生物科技股份有限公司,甘肃兰州730046 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《甘肃农业大学学报》2014年第3期32-36,共5页Journal of Gansu Agricultural University

基　　金：甘肃省科技计划资助(1104NKCA167)

摘　　要：根据Ovis aries interleukin(IL-2)序列设计1对引物,用PCR法扩增的目的基因进行克隆,将测序正确的IL-2基因片段和表达载体pPIC9K同时双酶切后相连,构建pPIC9K-IL-2重组表达载体.将线性化的载体pPIC9K-IL-2电转化毕赤酵母GS115,对重组酵母转化子经MD平板筛选和PCR分析鉴定后,用G418(4g/L)筛选到多拷贝菌株,进行不同条件下甲醇诱导表达.结果表明:经PCR鉴定IL-2基因已整合到酵母基因组中,表达产物经SDS-PAGE电泳分析发现相对分子质量约为18ku目的蛋白表达条带,本研究成功构建了高效表达重组羊IL-2毕赤酵母菌株,为新型细胞因子佐剂的研发奠定了基础.A pair of primers was designed to clone IL-2 gene according to the Ovis aries interleukin-2（IL-2）sequence and the recombinant expressed vector pPIC9K-IL-2 was constructed.The recombinane plasmid was transformed into Pichia pastoris GS115by electroporation.The transformants were selected with MD culture plate and identified by PCR.The multicopy recombinant P.pastoris strain was selected by G418（4g/L）resistance.The result showed that IL-2 gene was integrated with chromosome of P.pastoris by PCR identification.The expressed product had a molecular weight of 18ku band by SDS-PAGE analysis.The recombinant P.pastoris GS115 was successfully constructed,which provided the basis for research and development of a novel cytokine adjuvant.

关 键 词：绵羊 白介素-2基因 毕氏酵母 基因表达 SDS-PAGE 

分 类 号：S852.4[农业科学—基础兽医学]