不同复性方法制备的rhBMP

作者：赵玮钦，陈苏民，王涛，张晓楠，王丽

【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

　　Comparison of different refolding methods in inducing ectopic bone formation by rhBMP2m

　　【Abstract】 AIM: To refold recombinant human bone morphogenetic protein2 mature peptide (rhBMP2m) by different methods, and compare the activity of inducing the formation of ectopic bone. METHODS: The engineered strain, which could express rhBMP2m at a high level, was fermented in high density, and induced by temperature. The bacterial cells were collected and lyzed. The gained inclusion bodies were purified by ionexchange chromatography. The purified rhBMP2m was refolded by three different methods: ① rhBMP2m was dialyzed with pH 4.8 buffer for refolding. ② rhBMP2m was refolded by means of dilution method in pH 9.0 buffer. ③ rhBMP2m 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 rhBMP2m was 98% after ionexchange chromatography. ① The resulting rhBMP2m 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 rhBMP2m was planted into mouse muscles, it induced ectopic cartilage after 2 weeks and trabecular bone after 4 weeks. ② rhBMP2m was soluble after refolding with pH 9.0 buffer. The refolded rhBMP2m seemed dissolvable by macroscopic observation, but lost over 90% after filtrating sterilization.After 1 mg of this lyophilized rhBMP2m 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, rhBMP2m was precipitated. When 1 mg of insoluble rhBMP2m was planted into muscles, it induced trabecular bone in 2 weeks and myeloid cells after 4 weeks. CONCLUSION: Compared with acid and alkaline condition, rhBMP2m refolded in neutral condition has the best activity of inducing the formation of bone, but rhBMP2m refolded in acid condition has better dissolubility.

　　【Keywords】 human bone morphogenetic protein2 mature peptide; high density fermentation; protein purification; protein refolding

　　【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(rhBMP2m)，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rhBMP2m用三种不同方法复性：①对pH 4.8复性液透析复性；②对pH 9.0复性液稀释复性；③对pH 6.5复性液透析复性. 不加任何载体，1 mg复性后rhBMP2m直接植入小鼠肌肉观察诱骨生成. 结果：得到纯度为98％的rhBMP2m. ①pH 4.8透析复性后蛋白可溶. 经过滤除菌，可溶性rhBMP2m没有损失. 冻干后植入肌袋，2 wk后诱导生成软骨，4 wk诱导生成骨小梁. ②pH 9.0稀释复性后虽然肉眼观察蛋白似为可溶，但经除菌膜过滤rhBMP2m损失90%以上. 冻干后植入肌袋，1 wk诱导生成软骨，2 wk诱导软骨内成骨，3 wk出现骨小梁. ③pH 6.5透析复性后蛋白不可溶，植入肌袋，2 wk诱导生成骨小梁，4 wk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rhBMP2m的诱骨活性最好，但酸性条件下复性的rhBMP2m溶解度好.

　　【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

　　0　引言

　　重组人骨形成蛋白2成熟肽(recombinant human bone morphogenetic protein2 mature peptide, rhBMP2m)，属于转化生长因子β（transforming growth factorβ, TGFβ）超家族，它在骨骼再生与修复过程，以及胚胎发育的不同阶段发挥着重要的作用［1］. 目前，rhBMP2m已经被商业化生产并应用在多种临床研究中，包括治疗骨质缺损、颌面修复等. 利用哺乳动物细胞制备rhBMP2m，其表达水平很低,纯化过程复杂，使得产品价格高昂，制约着其推广应用. 利用大肠杆菌表达rhBMP2m，获得包含体，体外复性后可以得到具有生物活性的rhBMP2m［2］. 但是其复性过程复杂，总收得率低，复性缓冲液所要试剂昂贵. 本研究采用三种不同的复性方法，通过动物试验比较不同方法复性的rhBMP2m的诱骨活性，为获得一种简单而且廉价的方法以改进活性rhBMP2m的生产.

　　1　材料和方法

　　1.1材料

　　雄性健康昆明小鼠12只，体质量18~22 g，由第四军医大学实验动物中心提供；溶菌酶，DNaseⅠ购自美国Invitrogen公司；菌种DH5α/pDH2rhBMP2m，BCA蛋白定量试剂为第四军医大学生物化学与分子生物学教研室配制并保存；低分子量蛋白marker购自上海生化所； 15 L全自动发酵罐购自美国NBS公司；SPSepharose FF，QSepharose FF购自瑞典Pharmacia公司；低分子蛋白透析膜（截留分子量10 000）购自北京华美公司；超声粉碎器购自美国Cole Parmer Instrument公司；低温高速离心机购自湖南湘西仪器仪表厂；MinitanTM超滤系统购自美国MILIPORE公司；SDSPAGE垂直平板电泳系统购自美国BIORED公司；冻干机购自丹麦HETO公司； 0.22 μm微孔滤膜来自上海兴亚净化材料厂.

　　1.2方法

　　1.2.1DH5α/pDH2rhBMP2m

　　高密度发酵、裂菌、包涵体洗涤取-70℃保存的DH5α/pDH2rhBMP2m工程菌株甘油菌种， 划LB琼脂平板， 含100 mg/L氨苄青霉素， 32℃培养20 h. 挑单菌落入6 mL LB，30℃摇床培养16 h. 转入300 mL LB，30℃摇床培养10 h. 转入15 L发酵罐30℃培养,计算机自动控制搅拌、通气溶氧，30℃培养16 h. 发酵罐内42℃诱导表达4 h，结束发酵. 裂解缓冲液(100 g/L蔗糖，10 mmol/L pH 8.0 TrisHCL，1 mmol/L EDTA)悬浮菌体，加溶菌酶，冰浴中超声裂菌，离心后收得包涵体. 洗涤液(5 g/L TritonX100，10 mmol/L pH 8.0 TrisHCl，1