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猪IL-2基因真核表达载体的构建及在酵母中的表达
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张巧颖1, 2，景志忠1，栗震亚2 ，陈国华1
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(1.中国农业科学院 兰州兽医研究所 甘肃省动物寄生虫病重点实验室 家畜疫病病原生物学国家重
点实验室，甘肃 兰州730046； 2.兰州大学 生命科学学院，甘肃 兰州730000）
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摘要：
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用真核表达引物从pGEM-IL-2重组质粒中扩增出猪IL-2基因，将目的基因和真核表达载体pPIC9K连接转入E.coli的JM109中，得到了猪pPIC9K-IL-2重组表达质粒。通过电激法将经SalⅠ酶切线性化的pPIC9K-IL-2质粒转化到巴斯德毕赤酵母GS115感受态细胞中，利用甲醇诱导表达，经SDS-PAGE电泳分析，表明在摇床水平及发酵罐中均表达出约17ku大小的分泌性目的蛋白，采用Sephadex G-100分子筛层析对其表达产物进行纯化，纯化结果理想。 |
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关键词：
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白细胞介素-2；基因；毕赤酵母；表达 |
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中图分类号：Q 786 文献标识码：
A 文章编号：1673-4696(2006)04-0315-05 |
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Construction of eukaryotic vector with porcine IL-2 gene
and its expression in Pichia pastoris |
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ZHANG Qiao-ying1，2，JING Zhi-zhong1，LI
Zhen-ya2，CHEN Guo-hua1 |
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(1.Key Laboratory of Veterinary Parasitology of Gansu Province/State Key Laboratory of Veterinary Etiological
Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences, Lanzhou 730046,China;
2.School of Life Science, Lanzhou University, Lanzhou 730000,China) |
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Abstract: The porcine IL-2 gene, which was amplified from pGEM-IL-2 with expression primers, was inserted into the expression vector pPIC9K and then transformed into Escherichia coli. Positive recombinants pPIC9K- IL-2 were selected,sequenced and linearized by SalⅠ. After the linearized DNA of pPIC9K- IL-2 was transformed into Pichia pastoris GS115 by electroporation, the IL-2 gene was expressed by induction of methyl alcohol. SDS-PAGE analysis showed that the IL-2 gene was expressed successfully in Pichia pastoris both in the shake culture and the fermentor, and the porcine IL-2 protein of approximately 17ku in size was secreted. The expressed protein was successfully by using Sephadex G-100. |
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Key words: IL-2；gene；Pichia pastoris; expression |
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