| 产人胰岛素毕赤酵母工程菌的构建 |
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| 引用本文： | 王隆飞,方园园,陈功,博涛,侯健华,杨少辉,李桂兰,李欣,李明刚.产人胰岛素毕赤酵母工程菌的构建[J].南开大学学报,2006,39(5):69-73. |
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| 作者姓名： | 王隆飞 方园园 陈功 博涛 侯健华 杨少辉 李桂兰 李欣 李明刚 |
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| 作者单位： | 南开大学生命科学学院 天津300071 |
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| 基金项目： | 国家高技术研究发展计划(863计划) |
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| 摘 要： | 根据人胰岛素的氨基酸序列和酵母偏爱的密码子,采用基因半合成技术合成人胰岛素基因.将合成的胰岛素基因克隆到pP IK 9质粒,构建了毕赤(P ich ia)酵母重组表达载体pP IN S319,用B g lⅡ线性化后用电转化法导入毕赤酵母G S ll5菌株,经过营养筛选和PCR复筛,得到含人胰岛素基因的毕赤酵母工程菌株.经SDS-PAGR和密度扫描分析表明,合成的人胰岛素基因能够在毕赤酵母中高效分泌性表达,表达量可达0.563 m g/mL,表达产物无需转肽过程,只经过胰蛋白酶和羧肽酶B简单处理即得到优质重组人胰岛素,其体内活力约为30 IU/m g.
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| 关 键 词： | 人胰岛素基因 毕赤酵母 分泌性表达 |
| 文章编号： | 0465-7942(2006)05-0069-05 |
| 收稿时间： | 09 10 2005 12:00AM |
| 修稿时间： | 2005年9月10日 |
Construction of a Transgenic Pichia Pastoris Strain Yielding Human Insulin |
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| Wang Longfei,Fang Yuanyuan,Chen Gong,Bo Tao,Hou Jianhua,Yang Shaohui,Li Guilan,Li Xin,Li Minggang.Construction of a Transgenic Pichia Pastoris Strain Yielding Human Insulin[J].Acta Scientiarum Naturalium University Nankaiensis,2006,39(5):69-73. |
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| Authors: | Wang Longfei Fang Yuanyuan Chen Gong Bo Tao Hou Jianhua Yang Shaohui Li Guilan Li Xin Li Minggang |
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| Institution: | College of Life Sciences, Nankai University, Tianjin 300071 , China |
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| Abstract: | Insulin gene was synthesized according to the amino acid sequence of human insulin and yeast preferential codons.The insulin gene was cloned into pPIC9 vector and expression vector pPINS319 was constructed.The expression vector was digested with BgllI and then used to transform Pichia Pastoris GSll5 by electroporation.The expression analysis showed that the insulin gene was able to expressed efficiently in Pichia Pastoris.SDS-PAGE and density scanning analysis indicated that the highest expression amout was about(0.563 mg/mg.) After treatment by trypsin and carboxypeptidase B,the endo-bioactivity of the recombinant insulin obtained was approximately 30 IU/mg. |
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| Keywords: | human insulingene Pichia Pastoris secretory expression |
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