Green I R, Fiskerstrand C, Bertoni G, Roy D J, Peterhans E, Sargan D R
Department of Veterinary Pathology, University of Edinburgh, UK.
Cytokine. 1993 May;5(3):213-23. doi: 10.1016/1043-4666(93)90007-r.
We have expressed and partially purified recombinant ovine tumour necrosis factor alpha (rovTNF-alpha) using a yeast Ty, virus like particle, expression system. RovTNF-alpha is at least as active as recombinant human TNF-alpha (rhTNF-alpha) in two different bio-assays performed on ovine material, whilst approximately 1000-fold more rovrTNF-alpha than rhTNF-alpha is required to induce the same level of cytotoxicity in TNF-sensitive murine cell lines L929 and WEHI 164 clone 13. When cytotoxic assays are performed on the porcine TNF sensitive cell line PK(15)-1512, rovTNF-alpha shows about 2 logs greater activity than on murine cells, whilst rhTNF-alpha is about 1 log more active. A monoclonal antibody, raised against rovTNF-alpha, has been used to demonstrate the presence of nanogram amounts of an appropriately sized glycoprotein to be native ovine TNF-alpha in supernants of LPS stimulated ovine alveolar macrophages. These samples show no detectable cytotoxicity to L929 cells, although they show activity attributable to TNF-alpha (through neutralization by a polyclonal antiserum raised to rovTNF-alpha) in an assay on ovine material. The relative lack of activity on murine cells helps to explain previous reports of inability to assay native ovine TNF-alpha using these cells, in spite of their routine use to assay TNF-alpha from several other species. The sequence features in ovine TNF-alpha which might reduce its affinity for the murine TNF type 1 receptor are discussed.
我们使用酵母Ty病毒样颗粒表达系统表达并部分纯化了重组羊肿瘤坏死因子α（rovTNF-α）。在对羊材料进行的两种不同生物测定中，rovTNF-α的活性至少与重组人TNF-α（rhTNF-α）相同，而在TNF敏感的小鼠细胞系L929和WEHI 164克隆13中，诱导相同水平的细胞毒性所需的rovTNF-α比rhTNF-α多约1000倍。当在猪TNF敏感细胞系PK(15)-1512上进行细胞毒性测定时，rovTNF-α在该细胞系上的活性比在小鼠细胞上高约2个对数，而rhTNF-α的活性则高约1个对数。一种针对rovTNF-α产生的单克隆抗体已被用于证明在脂多糖刺激的羊肺泡巨噬细胞上清液中存在纳克量的大小合适的糖蛋白，该糖蛋白为天然羊TNF-α。这些样品对L929细胞没有可检测到的细胞毒性，尽管在对羊材料的测定中它们显示出可归因于TNF-α的活性（通过用针对rovTNF-α产生的多克隆抗血清中和）。在小鼠细胞上相对缺乏活性有助于解释先前关于无法使用这些细胞测定天然羊TNF-α的报道，尽管它们通常用于测定来自其他几个物种的TNF-α。本文讨论了羊TNF-α中可能降低其对小鼠TNF 1型受体亲和力的序列特征。
