骨形成蛋白10成熟肽在大肠杆菌中的表达纯化及活性分析  

Expression and Purification of Mature Bone Morphogenetic Protein 10 in Escherichia coli and its Activity Analysis

作　　者：李文军[1] 郑雄飞[1] 龚笑海[1] 金坚[1] 

机构地区：[1]江南大学药学院,江苏无锡214122

出　　处：《生物技术通讯》2016年第4期488-491,共4页Letters in Biotechnology

摘　　要：目的:以生物制备骨形成蛋白10(BMP10)为目标,研究BMP10成熟肽在大肠杆菌中的表达及活性。方法:以人源BMP10成熟肽基因为模板,PCR获得N端带有组氨酸标签(6×His)的融合基因6×His-m BMP10,构建p ET28a/m BMP10表达载体;热击转染大肠杆菌BL21(DE3)菌株,卡那霉素抗性筛选获得重组表达菌株BL21/p ET28a-6×His-m BMP10,IPTG诱导表达后利用SDS-PAGE电泳、Western印迹对蛋白进行分析;超声波破碎菌体,收集包涵体,镍柱亲和层析纯化获得电泳纯目的蛋白;透析复性后,非还原SDS-PAGE检验目标蛋白的二聚体形成;通过体外细胞实验检测蛋白活性。结果:纯化得到纯度90%以上的m BMP10,复性后二聚体得率约为40%;活性实验测得P19细胞的Smad6蛋白表达上调3倍左右。结论:通过大肠杆菌表达体系获得具有生物活性的BMP10,为后续作用机理研究奠定了实验基础。Objective： In biological preparation of bone morphogenetic protein 10（BMP10） as the goal, researchexpression and activity of BMP10 mature peptide in Escherichia coli. Methods： With mature anthropogenic BMP10 gene as a template, PCR to obtain the fusion gene 6×His-m BMP10 with N-terminal histidine labels（6×His）, con-struct p ET28a/BMP10 expression vector; heat stroke transfection E.coli BL21（DE3） strains, kanamycin resistancescreen to obtain recombinant expression strains BL21/p ET28a-6×His-m BMP10. After IPTG induced, analysis pro-teins by using SDS-PAGE electrophoresis and Western blotting. Bacteria ultrasonic broken, collect inclusions, nick-el column affinity chromatography purification for electrophoresis pure of purpose protein. After dialysis renatur-ation, the non-reducing SDS-PAGE found target protein to form dimers. In vitro cell activity detection, tested pro-tein activity at last. Results： Purity more than 90% of the purified m BMP10, dimers after renaturation yieldaround 40%. Active experiment measured the Smad6 protein expression of P19 cells increase about three times.Conclusion： Through the E.coli expression system to obtain bioactive BMP10, laid the experimental foundation forsubsequent mechanism study.

关 键 词：骨形成蛋白10 包涵体 亲和层析 透析复性 活性检测 

分 类 号：Q78[生物学—分子生物学]