摘要
利用PCR方法,将经RACE方法克隆到的中药青蒿鲨烯合酶cDNA(AF302464)开放阅读框的3′末端截短99bp,插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间,构建N端和C端均携带有HIS6表达标签的鲨烯合酶重组表达载体pETSSA.将pETSSA转入大肠杆菌BL21(DE3),0.5mmol/LIPTG(isopropyl-beta-D-thiogalactoside)28℃诱导重组鲨烯合酶的表达.表达产物经镍琼脂糖柱纯化.纯化蛋白加入酶促反应体系(含FPP和NADPH),GC-MS分析酶促反应体系的正己烷萃取物,结果显示重组鲨烯合酶可以催化FPP向鲨烯的转化.青蒿鲨烯合酶的功能鉴定为进一步利用反义或RNAi技术限制甾类生物合成,从而提高青蒿中的青蒿素含量提供了基础.
A 99 bps 3＇end truncated open reading frame of cDNA encoding squalene synthase （AF302464） from Artemisia annua was subcloned into a bacterial expression vector pET30a（ ＋ ） in frame with N-terminal and C-terminal His6-tag using Nco Ⅰ and BamH Ⅰ , and recombinant vector pETSSA was then introduced into Escherichia coli strain BL21 （DE3）. Recombinant squalene synthase was induced at 28 ℃ by adding 0. 5 mmol/L IPTG （isopropyl-beta-D-thiogalactoside） and purified using immobilized metal affinity chromatography on Ni^2＋ columns. GC - MS analysis showed that the purified recombinant squalene synthase could catalyze the formation of squalene from FPP in the presence of NADPH. This work lays the foundation of highly producing artemisinin from A. annua using antisense or RNAi methods to inhibit the squalene synthase. Fig 5, Ref 15
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2007年第3期309-312,共4页
Chinese Journal of Applied and Environmental Biology
基金
国家自然科学基金项目(No.30371740)资助~~
关键词
鲨烯合酶
大肠杆菌表达
功能鉴定
青蒿
squalene synthase
Escherichia coli expression
functional identification
Artemisia annua L.
