MOLECULAR-CLONING AND CHARACTERIZATION OF TUMOR-NECROSIS-FACTOR-ALPHA(TNF-ALPHA) FROM THE AUSTRALIAN COMMON BRUSHTAIL POSSUM, TRICHOSURUS-VULPECULA

Immune responses in the Australian common brushtail possum (Trichosuru s vulpecula) and in particular the role of cytokines are poorly unders tood. We have undertaken to isolate cytokine genes using reverse trans criptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha. Primers were designed from consen sus sequences at the N-terminus end of eutherian mammalian TNF-alpha a nd the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleot ide sequence of the protein coding region of this cDNA shared 66-69% i dentity with other mammalian TNF-alpha genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-a lpha. Possum TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared fr om transformants and the purified GST/TNF-alpha fusion protein exhibit ed cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L92 9 cells and stimulated proliferation of possum thymocyte cells. The in duction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers. Macrophages culture d in the presence of LPS showed enhanced transcription of TNF-alpha mR NA. This is the first report of the cloning and sequence analysis of t he cDNA encoding a marsupial cytokine gene.