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在用大肠杆菌表达人骨形成蛋白一3羧基端肽段(rhBMP-3C)获得成功的基础上,进一步探讨rhBMP-3C的纯化方法.方法:采用酶溶法裂菌释放出包涵体,继用包涵体洗涤液及5mol/L以下脲溶液连续洗涤,可获得较高纯度的包涵体,然后经透析缓慢除去变性剂,并用低浓度的硫酸铜和精氨酸促进复性,可获得较好的复性效果,采用离子交换层析法对复性处理后及变性状态下的rhBMP-3C进行分离纯化.结果:两种条件下表现出不同的洗脱特性,但SDS-PAGE鉴定结果均呈单一的蛋白带,小鼠肌肉植入试验表明,纯化复性后的rhBMP-3C具有明显的诱骨活性.结论:本实验为rhBMP-3C的大规模生产和临床应用创造了条件.
Based on the successful expression of human bone morphogenetic protein-3 carboxyterminal peptide (rhBMP-3C) in Escherichia coli, the purification of rhBMP-3C was further explored. Methods: Inclusion bodies were released by enzymatic lysozyme, followed by continuous washing with inclusion body washing solution and urea solution of 5 mol / L to obtain higher purity inclusion bodies. The denatured agent was slowly removed by dialysis and treated with low concentration of Copper sulfate and arginine can promote renaturation, and obtain better renaturation effect. The rhBMP-3C was purified by ion exchange chromatography after denaturalization and denatured. Results: Different elution characteristics were observed under the two conditions. However, the results of SDS-PAGE showed a single protein band. Muscle implantation experiments showed that the purified rhBMP-3C had obvious osteoinductive activity. Conclusion: This experiment created the conditions for large-scale production and clinical application of rhBMP-3C.