| GAPDH基因克隆及其作为毕赤酵母内标物的可靠性研究 |
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| 引用本文： | 张儒,张变玲,谢涛,李谷才,唐勇军,蒋开军.GAPDH基因克隆及其作为毕赤酵母内标物的可靠性研究[J].生物技术通报,2011(2). |
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| 作者姓名： | 张儒 张变玲 谢涛 李谷才 唐勇军 蒋开军 |
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| 作者单位： | 1. 湖南工程学院化学化工学院,湘潭,411104
2. 第四军医大学唐都医院疼痛生物医学研究所,西安,710038 |
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| 基金项目： | 湖南省自然科学基金项目(07JJ6031) |
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| 摘 要： | 为了测定GAPDH在巴斯德毕赤酵母中作为内参蛋白的有效性,利用PCR鉴定巴斯德毕赤酵母中GAPDH基因,分别在含葡萄糖、甲醇、甘油培养基及不同温度下培养巴斯德毕赤酵母,采用SDS-PAGE、Western blotting和催化活性检测。测序结果与已报道的巴斯德毕赤酵母GAPDH基因完全一致。SDS-PAGE结果显示,表达蛋白分子量为35.4 kD,与预期分子量相符。Western blotting和催化活性测定显示,在葡萄糖诱导下GAPDH表达最高,甲醇次之,甘油最低。37℃下GAPDH表达略高于28℃。在同一诱导物或温度下,GAPDH表达不会随浓度或培养时间发生明显变化。结果表明,在同一种诱导方式下,GAPDH可以作为异源蛋白表达的内参对照。
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| 关 键 词： | GAPDH 巴斯德毕赤酵母 内参对照 Western blotting |
Cloning of GAPDH Gene of Pichia pastoris and Its Reliability as Internal Control |
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| Zhang Ru,Zhang Bianling,Xie Tao,Li Gucai,Tang Yongjun,Jiang Kaijun.Cloning of GAPDH Gene of Pichia pastoris and Its Reliability as Internal Control[J].Biotechnology Bulletin,2011(2). |
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| Authors: | Zhang Ru Zhang Bianling Xie Tao Li Gucai Tang Yongjun Jiang Kaijun |
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| Institution: | Zhang Ru1 Zhang Bianling2 Xie Tao1 Li Gucai1 Tang Yongjun1 Jiang Kaijun1(1College of Chemistry and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,2Institute for Functional Brain Disorders,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038) |
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| Abstract: | It was to determine the validity of GAPDH as an internal protein for assessment in P.pastoris.The GAPDH gene from P.pastoris GS115 was identified by PCR.Expression of GAPDH gene in glucose,methanol,glycerol-grown cells at different temperature,was identified by SDS-PAGE,Western blotting and metabolic activity.The sequence analysis showed that the homology of the GAPDH gene with previously reported P.pastoris was 100%.The SDS-PAGE analysis revealed the protein with a molecular weight of 35.4 kD,and the Weste... |
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| Keywords: | Glyceraldehyde-3-phosphatedehydrogenase P pastoris Internal control Western blotting |
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