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本文旨在进一步了解重组人肿瘤坏死因子α(TNF-α)的生物学活性。首先在大肠杆菌中克隆出编码TNE-α的基因:裂菌,进而用一系列层析技术从不溶性物质中将TNF-α纯化至均一程度。重新折叠变性蛋白,该重新折叠的TNF-α在小鼠L929细胞上的溶细胞作用达1×10~7U/mg。4℃保存TNF-α。检测按Spofford的改良方法将小鼠L929(获自ATCC,CCl-1)或Hela细胞分别以12500或20000细胞/孔种于96孔平底微滴板上。培养24小时,加入TNF-α,继续培养48小时后,细胞溶解,用0.5%结晶紫溶液染色观察结果。对照细胞仅用培养基,以细胞溶解50%为1个单位。用标准细胞致病作用(CPE)测定抗病毒活性。用TNF-α处理Hela细胞,以脑心肌
This article aims to further understand the biological activity of recombinant human tumor necrosis factor alpha (TNF-alpha). First, a gene encoding TNE-α was cloned in Escherichia coli: fission cells, and then a series of chromatographic techniques were used to purify TNF-α from insoluble substances to a uniform extent. The renaturation protein was refolded, and the refolded TNF-α had a cytolytic effect of 1 × 10 -7 U / mg on mouse L929 cells. TNF-α was preserved at 4 ° C. Detection Mouse L929 (obtained from ATCC, CCl-1) or HeLa cells were seeded onto 96-well flat-bottomed microtiter plates at 12500 or 20000 cells / well, respectively, according to an improved method of Spofford. After culturing for 24 hours, adding TNF-α, culturing was continued for 48 hours, the cells were lysed and the results were stained with 0.5% crystal violet solution. Control cells only medium, cell lysis 50% to 1 unit. Antiviral activity was measured using standard cytopathic effect (CPE). Hela cells were treated with TNF- [alpha] in the myocardium