Tumor necrosis factor-alpha gene from mandarin fish, Siniperca chuatsi: molecular cloning, cytotoxicity analysis and expression profile.

In this report, we cloned a cDNA and the gene of mandarin fish (Siniperca chuatsi) tumor necrosis factor-alpha (TNF-alpha). The length of TNF-alpha cDNA was 1461 bp, contained an open reading frame (ORF) of 762 bp, which encoded 253 amino acids. The positions of cysteine residues, transmembrane sequence, and protease cleavage site were similar with other reported fish TNF-alpha and mammalian TNF-alpha. Mandarin fish TNF-alpha gene has a length of 1940 bp, and consists of four exons and three introns. There are three NF-kappaB/rel regulatory/binding sites in gene's 5' flanking region. Expression of mandarin fish TNF-alpha mRNA was constitutive in all tissues detected. After challenge of infectious spleen and kidney necrosis virus (ISKNV), which results a high mortality rate in mandarin fish and has caused huge economic losses in China, the expression of TNF-alpha mRNA in head kidney and kidney did not show significant changes from 0 day to 15 days post-infection. While in blood and spleen, the gene's mRNA showed up-regulations in 4 days post-infection and down-regulations in 15 days post-infection, which indicated the mRNA expression of TNF-alpha may related to the infection of ISKNV and the survival of mandarin fish. Mature form of recombinant mandarin fish TNF-alpha protein (400-4000 ng/ml) showed an evident apoptosis induction ability of human HeLa cells, in a dose-dependent manner, although to a lesser extent compared to the recombinant human TNF-alpha protein (200 ng/ml).