人肿瘤坏死因子及其突变体的基因工程下游工艺研究  

Downstream Technique Study of Human Tumor Necrosis Factor Alpha and its Mutant

作　　者：徐皓[1] 高长寿[1] 刘惠[1] 邓芳 潘华 陈常庆[1] 

机构地区：[1]中国科学院上海生物工程研究中心

出　　处：《生物工程学报》1998年第2期164-169,共6页Chinese Journal of Biotechnology

摘　　要：研究了重组人肿瘤坏死因子（ｒｈＴＮＦα）及其突变体［Ｌｙｓ２］人肿瘤坏死因子［Ｌｙｓ２］ｒｈＴＮＦα）的基因工程下游工艺。ｒｈＴＮＦα和［Ｌｙｓ２］ｒｈＴＮＦα的温控表达工程菌株在Ｂ．ＢｒａｕｎＥ１０型１５Ｌ自控罐中经发酵每升可得５０ｇ湿菌体。ｒｈＴＮＦα和［Ｌｙｓ２］ｒｈＴＮＦα的表达水平仍能保持在５０％以上，且表达产物为可溶性蛋白。所得菌体经超声破菌、硫酸铵沉淀，然后依次用ＤＥＡＥＳｅｐｈａｒｏｓｅＦＦ、ＣＭＳｅｐｈａｒｏｓｅＦＦ和ＳｅｐｈａｃｒｙｌＳ２００柱层析进行分离纯化，由每升发酵液菌体最终可得１ｇ左右的纯品，纯度达到９８％左右，ｒｈＴＮＦα的比活为～１５×１０８ＩＵ／ｍｇ，［Ｌｙｓ２］ｒｈＴＮＦα的比活为～６×１０８ＩＵ／ｍｇ。The study of down stream techniques for recombinant human necrosis factor alpha(rhTNFα) and its mutant(Lys 2)rhTNFα led to the results of approximately 50g wet recombinant E.coli per liter with high expression level(>50%) harvested from autocontrolled fedbatch culture in 15L fermentor(B.Braun).The rhTNFα and (Lys 2)rhTNFα expressed are totally soluble.Followed by the process of ultrasonication,ammonium sulfate precipitation,ionexchange chromatography(DEAESepharose FF,CMSepharose FF) and molecular sieve(Sephacryl S200),a yield of approximately 1g pure recombinant protein from 1L broth is obtained.The purity is up to 98%.The specific activity of rhTNFα and (Lys 2)rhTNFα are ～15×108 IU/mg and ～6×10 8IU/mg respectively.

关 键 词：肿瘤坏死因子 突变型 下游工艺 发酵 菌体破碎 

分 类 号：TQ464.7[化学工程—制药化工]