| 赵晓雅,李 婕,余 磊,郑桂兰,王洪钟.3β-羟基类固醇脱氢酶基因的克隆表达及其性质研究[J].现代生物医学进展英文版,2018,(2):201-204. |
| 3β-羟基类固醇脱氢酶基因的克隆表达及其性质研究 |
| Cloning and Expression of 3β - Hydroxy Steroid Dehydrogenase Gene and Its Properties Research |
| Received:May 23, 2017 Revised:June 18, 2017 |
| DOI：10.13241/j.cnki.pmb.2018.02.001 |
| 中文关键词: 3β-羟基类固醇脱氢酶 类固醇 酶活性 |
| 英文关键词: 3β-hydroxy steroid dehydrogenase Steroid Enzymatic activity |
| 基金项目:国家自然科学基金项目（21476124） |
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| 中文摘要: |
| 摘要 目的：构建3β-羟基类固醇脱氢酶（3β-HSD）异源表达系统，进一步探究其酶学性质。方法：通过分子生物学方法克隆来源于Mycobacterium neoaurum菌株的3β-羟基类固醇基因，构建重组质粒，运用HPLC方法检测酶反应体系的产物。结果：本实验构建了表达载体pet28a-hsd。优化诱导表达条件，发现3β-HSD异源表达的最适温度为16 ℃~25 ℃，37 ℃时蛋白表达为包涵体。此外，同一温度下不同IPTG浓度诱导的表达结果差异不大。利用纯化后的蛋白进行酶特性的研究，结果表明在3β-HSD酶反应体系中，30 ℃达到较高的酶活性；pH 7.5~9.0之间，酶活力较强，pH低于7.0时，酶活性明显下降；有机助溶剂DMSO对酶反应有抑制作用；Fe3+ 和Cu2+对酶反应有抑制作用。结论：本实验探究了分枝杆菌中3β-羟基类固醇脱氢酶的相关性质，为进一步研究该酶在类固醇代谢中的功能提供基础。 |
| 英文摘要: |
| ABSTRACT Objective: To construct a heterologous expression system of 3β-hydroxy steroid dehydrogenase, and to further explore its enzymatic properties. Methods: The 3β-hydroxy steroid gene derived from Mycobacterium neoaurum strain was cloned by molecular biology method. The recombinant plasmid was constructed and the product of enzyme reaction system was detected by HPLC. Results: The expression vector pet28a-hsd was constructed in this study. The optimal expression temperature of 3β-HSD was 16 ℃ ~ 25 ℃, and the protein was expressed as inclusion body at 37 ℃. In addition, the expression of different IPTG concentrations at the same temperature was not very different. The results showed that the enzyme activity was higher at 30 ℃ in the 3β-HSD enzyme reaction system, pH was between 7.5 and 9.0, the enzyme activity was stronger, the pH was lower than 7.0, the activity of the enzyme was decreased, Fe3+ and Cu2+ have an inhibitory effect on the enzyme reaction. Conclusion: This study explores the related properties of 3β-hydroxy steroid dehydrogenase in mycobacteria and provides a basis for further study of the function of the enzyme in steroid metabolism. |
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