摘要
[目的]利用简单快速的基因工程法来生产富含二硫键的芋螺毒素Mr VIB,寻找有效合成具有天然活性芋螺毒素的新途径。[方法]人工设计合成芋螺毒素Mr VIB基因引物来构建表达载体p ET22b(+)/His-Xa-Mr VIB,将其转化大肠杆菌BL21(DE3)plys S进行诱导表达。再利用Ni-NTA琼脂糖柱进行亲和层析纯化重组蛋白,Tricine-SDS-PAGE电泳分析重组蛋白表达形式。[结果]重组芋螺毒素His-Xa-Mr VIB(r His-Xa-Mr VIB)在大肠杆菌中获得有效分泌表达,经一步亲和层析获得纯度大于90%的重组芋螺毒素。[结论]基因工程方法能够有效分泌表达芋螺毒素Mr VIB,解决化学合成芋螺毒素产量低、成本高、难以纯化等问题。
[Objective]The disulfide-rich conotoxin Mr VIB was produced by simple and fast method of genetic engineering,to find new efficient ways to synthetic a natural active conotoxin. [Methods]Artificial design conotoxin Mr VIB gene was synthesized to construct expression vector p ET22b（＋）/His-Xa-Mr VIB,which was transformed into E. coli BL21（DE3） p Lys S and induced expression by IPTG. Recombinant proteins were purified by affinity chromatography using Ni-NTA agarose column,the expression and purification of recombinant proteins were analyzed by Tricine-SDS-PAGE. [Results]Recombinant conotoxin His-Xa-Mr VIB（r His-Xa-Mr VIB）was effectively secreted and expressed in E. coli,which was purified by one-step affinity chromatography and the purity of recombinant conotoxin was more than 90%. [Conclusion]The conotoxin Mr VIB was effectively secreted and expressed by genetic engineering method,which can solve the problem of chemical synthesis conotoxins that has low yield,high cost and difficult to purify.
出处
《生物技术》
CAS
CSCD
北大核心
2015年第1期66-70,共5页
Biotechnology
基金
海南医学院培育基金项目("基因工程法制备芋螺毒素Mr VIB"
No.HY2013-11)
海南医学院大学生创新课题("基因工程法制备芋螺毒素Mr VIB"
No.HYCX201327)
海南省自然科学基金项目("益智遗传多样性的AFLP分析"
No.814291)
海南医学院引进人才科研启动经费资助
关键词
芋螺毒素
大肠杆菌
基因工程
分泌表达
分离纯化
conotoxins
Escherichia coli
genetic engineering
secretory expression
separation and purification
