| 猪IL-2基因在毕赤酵母中的高效表达及生物活性分析 |
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| 引用本文： | 陈国华,贾怀杰,曾爽,房永祥,李小庆,景志忠,才学鹏.猪IL-2基因在毕赤酵母中的高效表达及生物活性分析[J].畜牧兽医学报,2011,42(4). |
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| 作者姓名： | 陈国华 贾怀杰 曾爽 房永祥 李小庆 景志忠 才学鹏 |
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| 作者单位： | 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 |
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| 基金项目： | 国家高新技术“863”项目(2006AA10A203); 国家自然科学基金项目(30871884) |
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| 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。
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| 关 键 词： | 猪IL-2基因 毕赤酵母 活性分析 |
Expression and Bioactivity Analysis of Porcine IL-2 in Pichia pastoris |
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| CHEN Guo-hua,JIA Huai-jie,ZENG Shuang,FANG Yong-xiang,LI Xiao-qing,JING Zhi-zhong,CAI Xue-peng.Expression and Bioactivity Analysis of Porcine IL-2 in Pichia pastoris[J].Acta Veterinaria et Zootechnica Sinica,2011,42(4). |
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| Authors: | CHEN Guo-hua JIA Huai-jie ZENG Shuang FANG Yong-xiang LI Xiao-qing JING Zhi-zhong CAI Xue-peng |
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| Institution: | CHEN Guo-hua,JIA Huai-jie,ZENG Shuang,FANG Yong-xiang,LI Xiao-qing,JING Zhi-zhong,CAI Xue-peng (Key Laboratory of Veterinary Parasitology of Gansu Province,Key Laboratory of VeterinaryPublic Health of Ministry of Agriculture,State Key Laboratory of Veterinary EtiologicalBiology,Lanzhou Veterinary Research Institute,Chinese Academy ofAgricultural Sciences,Lanzhou 730046,China) |
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| Abstract: | 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 SD... |
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| Keywords: | porcine interleukine 2 gene Pichia pastoris bioactivity analysis |
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