MOLECULAR-CLONING, EXPRESSION AND CHARACTERIZATION OF OVINE TNF-ALPHA

Tumour necrosis factor alpha (TNF-alpha) is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cell types. By hybridization with a human TNF-alpha cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNF-alpha encodes a polypeptide of 234 amino acids that, based on analysis of human TNF-alpha, is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to the equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin-D treated WEHI-164 cells and induce COS cells to produce and secrete interleukin 6 (IL-6). Further experiments demonstrated the importance of sequences within the 3' untranslated region of the cDNA in determining the level of expression of ovine TNF-alpha. Northern blot analysis was used to analyse the kinetics of induction of ovine TNF-alpha mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNF-alpha at 1 h and 5 h but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNF-alpha mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNF-alpha appears to exist as a single copy.