Seow H F, Rothel J S, Pepin M, David M J, Wood P R
CSIRO Division of Animal Health, Animal Health Research Laboratory, Parkville, Vic., Australia.
Vet Immunol Immunopathol. 1995 Feb;44(3-4):279-91. doi: 10.1016/0165-2427(94)05305-c.
Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 degrees C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-alpha from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L-1. Both rOvTNF-alpha and recombinant human TNF-alpha (rhTNF-alpha) exerted cytotoxicity on L929 cells. However, rOvTNF-alpha but not rhTNF-alpha stimulated proliferation of ovine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.
利用从脂多糖（LPS）刺激的肺泡巨噬细胞中分离的RNA，通过逆转录-聚合酶反应克隆了绵羊肿瘤坏死因子-α（OvTNF-α），所用引物基于人TNF-α cDNA序列。构建了一个携带绵羊TNF成熟形式编码序列的表达载体。重组Ov-TNFα（rOvTNF-α）表达为谷胱甘肽-S-转移酶（GST）融合蛋白。用凝血酶切割后产生不含GST部分的rOvTNF。在30℃的较低温度下培养以及使用大肠杆菌菌株AM207、AM305、E392和NM522，并没有显著提高rOvTNF-α从可溶部分的回收率。通过使用谷胱甘肽-琼脂糖亲和层析可快速简便地实现重组蛋白的纯化。在大肠杆菌JM109和AM207中获得的纯rOvTNF-α产量约为1mg/L。rOvTNF-α和重组人TNF-α（rhTNF-α）均对L929细胞具有细胞毒性。然而，rOvTNF-α而非rhTNF-α刺激了绵羊胸腺细胞的增殖。LPS刺激的绵羊肺泡巨噬细胞在刺激后约4小时检测到TNF-α mRNA表达的最高水平。
