胆固醇7,8位脱氢酶的表达及催化活性研究

doi:10.13523/j.cb.20170104

中国生物工程杂志

2017, Vol. 37

Issue (1): 21-26 DOI: 10.13523/j.cb.20170104

研究报告

胆固醇7,8位脱氢酶的表达及催化活性研究

张璟¹, 张文强¹, 秦慧民¹, 毛淑红¹, 薛家禄², 路福平¹

1. 天津科技大学生物工程学院工业发酵微生物教育部重点实验室天津 300457;
2. 河南利伟生物医药股份有限公司焦作 454800

Expression of Related Cholesterol Dehydrogenation Protein and Bioactivity Research

ZHANG Jing¹, ZHANG Wen-qiang¹, QIN Hui-min¹, MAO Shu-hong¹, XUE Jia-lu², LU Fu-ping¹

1. Key Laboratory of Industrial Fermentation Microbiology, National Engineering Laboratory for Industrial Enzymes(NELIE), College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China;
2. Henan Li Wei Medicine Co., Ltd, Jiaozuo 454800, China

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摘要：

果蝇的neverland基因是胆固醇7，8位脱氢的重要酶基因。为了探究其在胆固醇脱氢反应中的催化机制，将neverland分别克隆至表达载体pIEx-6及pXY212，再转染导入S2细胞并电转化到S. cerevisiae W303-1A中表达。Western blot结果证实NVD蛋白在重组S2细胞及S. cerevisiae W303-1A中实现了表达。胆固醇转化实验经HPLC分析发现，重组S2细胞可以将胆固醇转化为7-脱氢胆固醇，而重组S. cerevisiae W303-1A并不能实现胆固醇的7，8位脱氢。此外，在重组S. cerevisiae W303-1A和S2细胞的破碎液共同转化胆固醇及NVD体外转化实验中也未发现产物7-脱氢胆固醇的生成。实验结果显示，neverland基因在S2细胞中具有生物活性而在S. cerevisiae中没有生物活性，表明它在胆固醇脱氢时需要其它的伴侣蛋白协助，实验结果为进一步研究其催化机制提供了理论基础。

关键词： 胆固醇7; 8位脱氢酶; S2细胞; 酿酒酵母W303-1A; 生物转化; 7-脱氢胆固醇

Abstract:

The neverland from the Drosophila melanogaster play essential roles in cholesterol dehydrogenation. To clone the neverland from the Drosophila melanogaster cDNA library and construct the eukaryote expression plasmid pIEx-6-nvd and pXY212-nvd to expressing the recombinant NVD in S2 cell and S. cerevisiae W303-1A for further analyzing its bioactivity. Results of Western blot indicates the recombinant protein expressed by S2 cell and S. cerevisiae W303-1A can specifically combine with 6×His antibody. Results of HPLC shows the cholestrol can be converted to 7-dehydrocholesterol by the recombinant S2 cell, however, the purified NVD and the recombinant S. cerevisiae W303-1A can not conversion cholesterol to 7-dehydrocholesterol, even the mixed recombinant S. cerevisiae W303-1A cell lysate and S2 cell lysate, neither. As the above result shows, the NVD need some chaperonins to complete the electron-transfer reactions when mediates the cholesterol dehydrogenation reaction.

Key words: NVD 7-dehydrocholesterol S. cerevisiae W303-1A S2 cell Bioconversion

收稿日期: 2016-05-13 出版日期: 2017-01-25

ZTFLH:

Q819

基金资助:

国家“863”计划资助项目（2011AA02A211-06）

通讯作者: 路福平 E-mail: lfp@tust.edu.cn

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引用本文:

张璟, 张文强, 秦慧民, 毛淑红, 薛家禄, 路福平. 胆固醇7,8位脱氢酶的表达及催化活性研究[J]. 中国生物工程杂志, 2017, 37(1): 21-26.

ZHANG Jing, ZHANG Wen-qiang, QIN Hui-min, MAO Shu-hong, XUE Jia-lu, LU Fu-ping. Expression of Related Cholesterol Dehydrogenation Protein and Bioactivity Research. China Biotechnology, 2017, 37(1): 21-26.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170104 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I1/21

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