Primary structure of ovine tumor necrosis factor alpha cDNA

Abstract

The cytokine tumor necrosis factor alpha (TNFa) has been shown to be a potent immunoregulatory molecule involved in the pathogenesis of septic shock (1). Here, we report the nucleotide and deduced amino acid sequence of a sheep TNFa cDNA which was isolated from a Xgtl 1 sheep liver cDNA library using a 800 base pairs (bp)—complete coding region—human TNFa cDNA as a probe (Gontech). Following four consecutive high stringency screenings, a 1499 bp clone was isolated and double strand sequenced by dideoxy chain termination (Promega). The clone sTNF-2 (Figure 1) codes for the full length sheep TNFa protein as determined by comparison to the human TNFa gene (2). The 1499 bp cDNA has an open reading frame of 699 bp encoding a protein of 233 amino acids with a predicted molecular weight of 25408 Kda. By comparison to the amino acid sequence of human (3), mouse (4), and rabbit (5), it was deduced that the protein consists of a 76 amino acid leader sequence and a 157 amino acid mature protein. The amino acid sequence showed 88% homology to the human TNFa protein, contained the characteristic TNF family signature sequence (6), the highly conserved cysteine residues which are involved in the single disulfide bond in human TNFa, and one potential N-linked glycosylation site. The 3' untranslated region of the gene was found to include the highly conserved poly-AT region potentially involved in translational control (7), and the polyadenylation signal (8).