Green I R, Sargan D R
Department of Veterinary Pathology, University of Edinburgh, U.K.
Gene. 1991 Dec 30;109(2):203-10. doi: 10.1016/0378-1119(91)90610-n.
We have cloned and sequenced the ovine tumor necrosis factor-alpha (TNF-alpha)-encoding cDNA, using gene amplification by polymerase chain reaction (PCR) technology, to aid studies of assorted diseases in this species. We used primers selected from published TnfA sequences of other species on a cDNA template prepared from lipopolysaccharide-stimulated ovine alveolar macrophages, to generate a product representing the central region of the molecule. We then used a novel method based on 'inverse PCR' to generate a product containing the 5' and 3' ends of the molecule. Here, we present the complete sequence of the ovine TNF-alpha cDNA and compare it with other published TNF sequences. The cloned cDNA has a leader sequence of 156 bp followed by a protein-coding sequence of 702 bp and a 3'-untranslated region of 800 bp. The protein product of the gene is a protein of Mr = 25,586, 79% homologous to human TNF-alpha. An mRNA produced by alveolar macrophages, which hybridises to the cloned gene, is induced greatly, with a peak induction time of approx. 135 min, in response to stimulation by lipopolysaccharide and to plating on plastic. We also discuss the resolution of some artefacts of the inverse PCR technique.
我们利用聚合酶链反应（PCR）技术进行基因扩增，克隆并测序了绵羊肿瘤坏死因子-α（TNF-α）编码cDNA，以助力对该物种各类疾病的研究。我们在由脂多糖刺激的绵羊肺泡巨噬细胞制备的cDNA模板上，使用从其他物种已发表的TnfA序列中选取的引物，生成了代表该分子中心区域的产物。然后我们采用一种基于“反向PCR”的新方法，生成了包含该分子5'端和3'端的产物。在此，我们展示绵羊TNF-α cDNA的完整序列，并将其与其他已发表的TNF序列进行比较。克隆的cDNA有一个156 bp的前导序列，随后是一个702 bp的蛋白质编码序列和一个800 bp的3'非翻译区。该基因的蛋白质产物是一种Mr = 25,586的蛋白质，与人类TNF-α有79%的同源性。肺泡巨噬细胞产生的一种与克隆基因杂交的mRNA，在脂多糖刺激和接种到塑料培养皿上后，被极大地诱导，诱导峰值时间约为135分钟。我们还讨论了反向PCR技术一些假象的解决方法。
