不同复性方法制备的rhBMP-2m诱导异位成骨活性比较  

Comparison of different refolding methods in inducing ectopic bone formation by rhBMP-2m

作　　者：赵玮钦[1] 陈苏民[1] 王涛[1] 张晓楠[1] 王丽[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2007年第7期619-623,共5页Journal of the Fourth Military Medical University

摘　　要：目的:用三种不同方法复性重组人骨形成蛋白2成熟肽(rhBMP-2m),比较其异位诱骨活性.方法:将工程菌株进行高密度发酵、温度诱导表达,裂菌收集包涵体后经离子交换色谱分离纯化.纯化后的rhBMP-2m用三种不同方法复性:①对pH4.8复性液透析复性;②对pH9.0复性液稀释复性;③对pH6.5复性液透析复性.不加任何载体,1mg复性后rhBMP-2m直接植入小鼠肌肉观察诱骨生成.结果:得到纯度为98%的rhBMP-2m.①pH4.8透析复性后蛋白可溶.经过滤除菌,可溶性rhBMP-2m没有损失.冻干后植入肌袋,2wk后诱导生成软骨,4wk诱导生成骨小梁.②pH9.0稀释复性后虽然肉眼观察蛋白似为可溶,但经除菌膜过滤rhBMP-2m损失90%以上.冻干后植入肌袋,1wk诱导生成软骨,2wk诱导软骨内成骨,3wk出现骨小梁.③pH6.5透析复性后蛋白不可溶,植入肌袋,2wk诱导生成骨小梁,4wk后出现骨髓样细胞.结论:相比酸性和碱性条件,中性条件下复性的rh-BMP-2m的诱骨活性最好,但酸性条件下复性的rhBMP-2m溶解度好.AIM： To refold recombinant human bone morphogenetic protein-2 mature peptide （rhBMP-2m） by different methods, and compare the activity of inducing the formation of ectopic bone. METHODS： The engineered strain, which could express rhBMP-2m at a high level, was fermented in high density, and induced by temperature. The bacterial ceils were collected and lyzed. The gained inclusion bodies were purified by ion-exchange chromatography. The purified rhBMP-2m was refolded by three different methods ： ① rhBMP-2m was dialyzed with pH 4.8 buffer for refolding. ② rhBMP-2m was refolded by means of dilution method in pH 9.0 buffer. ③ rhBMP-2m was dialysed in pH 6.5 buffer to refold. It （ 1 mg） was planted into mouse muscles to observe the formation of bone without any carrier. RESULTS： The purity of the acquired rhBMP-2m was 98% after ion-exchange chromatography. ① The resulting rhBMP-2m was soluble after refolding with pH 4. 8 buffer and had no any loss after passing through sterilizing filter membrane. When 1 mg of the lyophilized rhBMP-2m was planted into mouse muscles, it induced ectopic cartilage after 2 weeks and trabecular bone after 4 weeks. ② rh- BMP-2m was soluble after refolding with pH 9.0 buffer. The refolded rhBMP-2m seemed dissolvable by macroscopic observation, but lost over 90% after filtrating sterilization. After 1 mg of this lyophilized rhBMP-2m was planted into muscles, cartilage appeared after 1 week, bone formed after 2 weeks, and trabecular bone was seen after 3 weeks. ③ After dialysis, rhBMP-2m was precipitated. When 1 mg of insoluble rhBMP-2m was planted into muscles, it induced trabecular bone in 2 weeks and myeloid cells after 4 weeks. CONCLUSION： Compared with acid and alkaline condition, rhBMP-2m refolded in neutral condition has the best activity of inducing the formation of bone, but rhBMP-2m refolded in acid condition has better dissolubility.

关 键 词：重组人骨形成蛋白2成熟肽 高密度发酵 蛋白质纯化 蛋白质复性 

分 类 号：R392.33[医药卫生—免疫学]