猪IL-2基因在毕赤酵母中的高效表达及生物活性分析  

Expression and Bioactivity Analysis of Porcine IL-2 in Pichia pastoris

作　　者：陈国华[1] 贾怀杰[1] 曾爽[1] 房永祥[1] 李小庆[1] 景志忠[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,农业部兽医公共卫生重点实验室,甘肃省动物寄生虫病重点实验室,兰州730046

出　　处：《畜牧兽医学报》2011年第4期551-556,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家高新技术“863”项目(2006AA10A203);国家自然科学基金项目(30871884)

摘　　要：本研究根据密码子偏爱原则设计猪IL-2引物,克隆其基因后构建pPIC9k-IL-2重组表达载体,线性化后电转化毕赤酵母GS115,获得优化密码子的重组酵母转化子;再用G418抗性梯度法筛选得到多拷贝重组菌株,不同条件下进行甲醇诱导表达。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为16、20ku两条目的蛋白表达带,脱糖基化分析表明目的蛋白得到了适度的糖基化修饰,Western blotting分析显示表达的蛋白具有良好的免疫反应性,分子筛纯化可以获得纯度为95%的蛋白质,淋巴细胞增殖活性分析表明所得的蛋白质具有促淋巴细胞增殖的活性。毕赤酵母表达系统可以高效地表达具有生物活性的重组猪IL-2蛋白分子。A pair of primers was designed to clone IL-2 gene according to the codon bias principle and the recombinant expressed vector pPIC9k-IL-2 was constructed.The recombinant plasmid was transformed into P.pastoris GS115 by electroporation,the positive recombinant strains was screened with different G418 concentration and induced with methanol.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 16 and 20 kD bands by SDS-PAGE analysis.The result of deglycosylation analysis indicated that the protein was glycosylated moderately.The protein was purified with sephadex column and had a bioactivity of lymphocyte proliferation.The Western blotting analysis showed that target protein had immunological activity.P.pastoris expressed system is an efficient way of production of pIL-2,the expressed product had a good bioactivity.

关 键 词：猪IL-2基因 毕赤酵母 活性分析 

分 类 号：S852.4[农业科学—基础兽医学]