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.
出处
《生物技术通讯》
CAS
2016年第4期488-491,共4页
Letters in Biotechnology
关键词
骨形成蛋白10
包涵体
亲和层析
透析复性
活性检测
bone morphogenetic protein 10
inclusion body
affinity chromatography
dialysis renaturation
activity detection
