大肠杆菌中芥蓝抗坏血酸过氧化物酶基因的高效表达  

Expression of ascorbate peroxidase gene of Chinese kale in E. coli

作　　者：黄加保[1] 唐蕾[1] 华子安[2] 毛忠贵[2] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2013年第7期1-5,共5页Food and Fermentation Industries

基　　金：国家自然科学基金资助项目(30972057)

摘　　要：将芥蓝抗坏血酸过氧化物酶(Apx)在大肠杆菌中高效表达。以芸薹属植物apx保守区基因序列为基础,利用PCR,RACE技术从芥蓝中扩增得到全长为753 bp的芥蓝apx编码基因,用DNAMAN V6软件分析该基因氨基酸组成,计算出蛋白分子质量约为27.6 kDa。通过构建重组质粒pET-28a-apx,转化大肠杆菌E.coli BL21,实现了芥蓝Apx的可溶性表达。通过单因素实验优化诱导表达条件,结果表明:0.2 mmol/L IPTG,23℃诱导培养10 h为最佳诱导条件。采用Ni2+亲和层析,纯化了重组蛋白,并得到重组酶最适反应条件为pH 6.5和40℃。为进一步研究Apx的生理特性和功能打下基础。In order to express ascorbate peroxidase （Apx） gene of Chinese kale in E. coli, we amplified apx coding sequence of Chinese kale by means of PCR and RACE based on the conserved sequence of ascorbate peroxidase gene in Brassica. Analyzed and calculated by DNAMAN V6, the molecular weight of recombinant protein was approximately 27.6 kDa. We constructed recombinant plasmid pET-28a-apx and transformed it into E. coli BL21. The Apx was expressed successfully in E. coli BL21 with the soluble form. The results from single-factor experiment showed that the optimal condition for Apx expression was induction at 23 ℃ for 10 hours with the addition of 0.2 mmol/L IPTG. The recombinant protein was purified by Ni^2＋ affinity chromatography and the best reaction condition for the enzyme was at pH6.5 and 40 ℃. The results provided the basis for the physiological and functional research of Chinese kale Apx.

关 键 词：芥蓝 抗坏血酸过氧化物酶 大肠杆菌 优化 

分 类 号：Q78[生物学—分子生物学]