| 马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达 |
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| 引用本文： | 韩云波,朱丽,张怡轩,魏维,戈梅. 马肝醇脱氢酶基因的克隆及其在大肠杆菌中的表达[J]. 生物学杂志, 2010, 27(2): 33-35. DOI: 10.3969/j.issn.1008-9632.2010.02.033 |
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| 作者姓名： | 韩云波 朱丽 张怡轩 魏维 戈梅 |
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| 作者单位： | 1. 沈阳药科大学生命科学与生物制药学院,沈阳,110016
2. 上海来益生物药物研发中心,上海,201203 |
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| 摘 要： | 利用RT-PCR技术从马肝扩增HLADH-E和HLADH-S基因,通过基因工程方法构建表达质粒pLY115E和pLY115S,在大肠杆菌中表达,并利用Ni柱分离纯化。利用紫外检测辅酶NADH在340nm的吸光值,来考察表达产物转化环己醇的活性。试验结果证明马肝醇脱氢酶HLADH-E和HLADH-S基因均能在大肠杆菌中表达,并且可溶性表达产物都具有氧化环己醇的活性,为马肝醇脱氢酶的进一步研究开发奠定了基础。
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| 关 键 词： | 马肝醇脱氢酶 克隆表达 活性测定 |
Cloning and expression of horse liver alcohol dehydrogenase gene in Escherichia coli |
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| HAN Yun-bo,ZHU Li,ZHANG Yi-xuan,WEI Wei,GE Mei. Cloning and expression of horse liver alcohol dehydrogenase gene in Escherichia coli[J]. Journal of Biology, 2010, 27(2): 33-35. DOI: 10.3969/j.issn.1008-9632.2010.02.033 |
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| Authors: | HAN Yun-bo ZHU Li ZHANG Yi-xuan WEI Wei GE Mei |
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| Affiliation: | HAN Yun-bo1,ZHU Li2,ZHANG Yi-xuan1,WEI Wei2,GE Mei2 (1.Life of Sciences , Bio-pharmaceutical College,Shenyang Pharmaceutical University,Shenyang 110016,2.Shanghai Health Creation Center of Biopharmaceuticals R&D,Shanghai 201203,China ) |
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| Abstract: | Two isoenzymes of horse liver alcohol dehydrogenase (HLADH) were cloned and expressed in Escherichia coli BL21 (DE3).The cDNAs for these isoenzymes of horse liver alcohol dehydrogenase were cloned from the mRNA of horse liver by RT-PCR technique.Both of the two isoenzymes of horse liver alcohol dehydrugenase were reconstructed into the vector pkk223-3 and expressed in Escherichia coli BL21 (DE3).The soluble recombinant horse liver alcohol dehydrogenases were purified by the Ni-NTA His ·Bind Resin,and the activities for these isoenzymes of horse liver alcohol dehydrogenase could be determined by the change of the concentration of NADH (β-nicotinamide adenine dinucleotide,reduced disodium salt,trihydrate).The change of NADH was detected by UV spectrophotometry at 340nm.Two obtained soluble isoenzymes for horse liver alcohol dehydrogenase could catalyze cyclobexanol,and the activity of the HLADH-E isoenzyme on cyclohexanol was higher than that of HLADH-S isoenzyme. |
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| Keywords: | horse liver alcohol dehydrogenase cloning and expression activity assay |
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