胆固醇7,8位脱氢酶的表达及催化活性研究  

Expression of Related Cholesterol Dehydrogenation Protein and Bioactivity Research

作　　者：张璟[1] 张文强[1] 秦慧民[1] 毛淑红[1] 薛家禄 路福平[1] ZHANG Jing ZHANG Wen-qiang QIN Hui-min MAO Shu-hong XUE Jia-lu LU Fu-ping(Key Laboratory of Industrial Fermentation Microbiology, National Engineering Laboratory for Industrial Enzymes （ NELIE）, College of Bioteehnology, Tianjin University of Science ＆ Technology, Tianjin 300457, China Henan Li Wei Medicine Co. , Ltd, Jiaozuo 454800, China)

机构地区：[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

出　　处：《中国生物工程杂志》2017年第1期21-26,共6页China Biotechnology

基　　金：国家"863"计划资助项目(2011AA02A211-06)

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p IEx-6及p XY212,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。The neverland from the Drosophila melanogaster play essential roles in cholesterol dehydrogenation. To clone the neverland from the Drosophila melanogaster c DNA library and construct the eukaryote expression plasmid p IEx-6-nvd and p XY212-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.

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 

分 类 号：Q819[生物学—生物工程]