Molecular cloning and characterization of tumour necrosis factor alpha (TNF‐α) from the Australian common brushtail possum, Trichosurus vulpecula

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Abstract
Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood. We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-. Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF- and the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF- was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF- genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-. Possum TNF- was expressed in both Saceharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared from transformants and the purified GST/TNF- fusion protein exhibited cytotoxic activity on the TNF--sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF- mRNA in alveolar macrophages was analysed by RT-PCR using possumspecific TNF- primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF- mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.