柱层析法纯化重组人肿瘤坏死因子α及其抑癌作用  

rhTNF-αpurification by colunm chromatography and its anti-tumor effects

作　　者：林丽珠[1] 陈春妹[1] 喻红[1] 吕群[1] 陈晓龙 姚锡惠 陈海明 赵寿元[1] 

机构地区：[1]复旦大学遗传学研究所

出　　处：《复旦学报（自然科学版）》1994年第5期553-558,共6页Journal of Fudan University：Natural Science

基　　金：上海市科学技术发展基金

摘　　要：半合成人肿瘤坏死因子ＴＮＦ－α在大肠杆菌中高表达．发酵后收集菌体经超声破碎，然后在１２０００ｒ／ｍｉｎ４℃离心１０ｍｉｎ，上清经６０℃热处理３０ｍｉｎ，离心上清用６０％饱和度的硫酸铰沉淀，再依次经过ＳｅｐｈａｄｅｘＧ—２５、ＤＥＡＥ—５２纤维素和ＳｅｐｈａｄｅｘＧ—７５柱层析，得到的纯化ｒｈＴＮＦ—α比活性为２×１０７ｕ／ｍｇ，回收率为２４％．经过ＳＤＳ—ＰＡＧＥ电泳和ＨＰＬＣ鉴定，纯度达９５％左右，分子量为１７×１０３．ｒｈＴＮ—α对热稳定性试验表明，—２０℃下贮存一年或者０℃下贮存一个月，ｒｈＴＮＦ—α活性仍保留５０％．人胃癌和肝癌细胞株的体外抑瘤试验和接种小鼠的活体抑瘤试验表明，本实验取得的ｒｈＴＮＦ—α具有明显的抑瘤生长作用．A recombinant human tumor necrosis factor (rhTNF) gene was highly expressed in E.Coli. The culture was then collected, ultrasonicated, and centrifuged at 12 000r/min at 4℃ for 10 min. The supernatant was heated at 60 ℃ for 30min and centrifuged. The supernatant was precipitated with 60% saturated ammonium sulfate and purified successively by column Sephadex G-25,DEAE-52 cellulose and Sephadex G-75. The biological specific activity of rhTNF-α is approximately 2.0×107 u/mg and recovery rate is 24%. The purity of the rhTNF-α preparation was shown to be 95% approximately by SDS-PAGE and HPLC.When stored at -20℃ for one year or at 0℃ for onemonth, it retains a biological activity of 50%. The rhTNF-α displayed cytostatic and cytolytic activities against several human tumor cell lines such as gastric carcinoma cell lines HGC-27 and MKN-28, and hepatoma cell line SMMC-7721 and also in vivo of mice.

关 键 词：肿瘤 坏死因子 柱层析 抑瘤试验 

分 类 号：R73－36[医药卫生—肿瘤]