大肠杆菌表达人新型肿瘤坏死因子的纯化

Purification of the new type human tumor necrosis factor expressed in E. coli

目的：制备符合临床要求的高纯度新型人肿瘤坏死因子（ｎｈＴＮＦ），建立简单、高效的纯化途径．方法：用本室构建的ｎｈＴＮＦ基因工程菌，经发酵、收菌、裂菌、硫酸铵盐析，Ｑ－Ｓｅｐｈａｒｏｓｅ及Ｓ－Ｓｅｐｈａｒｏｓｅ层析，得到高活性、高纯度的ｎｈＴＮＦ单一色谱峰，比活性达１×１０９Ｕ／ｍｇ蛋白．结果：ｎｈＴＮＦ纯度达９８％以上，活性回收率为７９％．结论：纯化方法简单，且达到较好效果，为ｎｈＴＮＦ的大规模制备和临床应用打下基础．

Objective: To prepare the high purified recombinant new type human tumor necrosis factor (nhTNF) with effective method. Methods: nhTNF expressed in E. coli was collected, disrupted and partially purified through ammonium sulfate preipitation. Then the crude sample was further purified by Q Sepharose, S Sepharose ion exchange chromatography sequentially. In SDS polyacrylamine gel electrophoresis the homogeneity of purified TNF was observed. Its specific activity reached 1.1×10 9 U/mg protein. Result: The purity of nhTNF was more than 98%, and the recovery rate 79%. Conclusion: A simple and effective purification method for nhTNF is established. It is the basis for large scale preparation and clinical application of the nhTNF.