重组人骨形态发生蛋白-2在大肠杆菌中的表达及纯化

Expression and purification of recombinant bone morphogenetic protein-2 in E.coli

目的用基因工程技术在大肠杆菌中表达人骨形态发生蛋白-2(hBMP-2)。方法hBMP－2原核表达载体pYR(pBV220－hBMP-2)转化 E coli BL21,SDS－PAGE分析工程菌活化状态以及诱导时间与目的蛋白表达率的关系。离子交换层析 DEAE和分子筛 S－300纯化重组蛋白,自然缓降复性法对其复性。结果SDS－PAGE显示在相对分子质量约为13000时出现明显外源蛋白表达带,而且当工程菌30℃活化至D600约为0．45时,其表达效率较高;在此状态下,温度诱导衣达4h,目的蛋白表达量最高,以后随着时间的延长,表达量稍下降。重组蛋白经纯化后植人小鼠肌肉,组织学观察到肌肉内大量间允质细胞增生以及软骨与骨形成。结论重组hBMP－2具有良好异位成骨活性。

Objechve To explore the method for producing human bone morphogenetic protein-2 (hBMP-2) by gene engi-neering techniques. Methods E.coli BL21 was thensformed with recombinant plasmid pYR (pBV220-hBMP-2) under different conditions, and SDS-PAGE analysis was conducted to observe the effects of the activation status and induction time of thebacterium on the target protein expression. The inclusion bodies obtained from E.coli were purified by anion exchange chro-matography DEAE and molecular sieve S-300, and the recombinant protein was renathed by dialyse. Results SDS-PAGEanalysis showed a conspicuous band after induction signilerg a new fOreign protein with relativc molecular mass of approxi-mately 13 000. After activation of the bacteria when D600 was about 0.45, most efficient expression of rhBMP-2 was achievedwhich reached the peak 4 h after induction with heat. Implantation of the purified recombinant hBMP-2 resulted in prolifera-tion of mesenchymal cells and new cartilage and bone formation, as shown by histological analysis 4 weeks after implantation.Conclusion hBMP-2 produced by gene engineering techniques possesses the biological capacity of ectopic bone formation.