Molecular Cloning Of CDNA Encoding Tumor Necrosis Factor Alpha (TNFα) In Black Sea Bream (Acanthopagrus Schlegeli) And Its Expression In Vitro

Based on homology strategy, the full length cDNA coding tumor necrosis factor alpha (TNFa) was cloned in black Sea bream using a pair of degenerated primers according to the conserved regions of other animal TNFa genes. The cDNA of BTNF had 1288bp nucleotides including a short 5' untranslated region UTR of 38bp, 3' UTR of 491bp and an open reading frame (ORF) of 759bp that could encode a propeptide of 253 amino acids with putative MW of 28kDa and putative PI of 4.94. In 3' UTR, there were several mRNA instability motifs and three endotoxin- response-motifs which should be involved in TNFa gene regulation. The deduced peptide belonged to membrane protein which had a transmembrane domain, but lacked clearly N-glycosylation site and signal peptide. The BTNF amino acids sequence shared high identity with piscine TNFa, mammalian TNFa and TNF homolog by identity and similarity research. Phylogentic analysis show that the piscine TNFa was located independently in another different branch compared with mammalian TNFa and TNF InterPro analysis indicate that the peptide included a TNF2 profile and a TNF family signature. The secondary structure analysis show that the BTNF was similar to mammalian TNFa, which had two conserved cysteine sites forming a disulphide bridge and conserved in the sites which were associated to form biological activity trimeric proteins. Tertiary analysis reveal that the BTNFa was similar to mammalian TNF which were sandwich structures containing two stacked pleated sheets each formed by five anti-parallel (3 strands that adopted a classical 'jelly-roll' topology. The structure analysis deduce that the BTNF should be TNFa, not TNF. RT-PCR reveal that the TNF transcript was constitutively expressed in black seabream, and the BS TNF transcript could be detected in most of the detected tissue both in control and stimulated fish, but the level of the expression was different, the BS TNFa were mainly expressed in head kidney, spleen and gill, whenas the faint expression can be detected in blood, brain, heart, liver and kidney.In order to study the function of the TNFa, TNFa mature peptide region was subcloned into pQE30 to construct the recombinant TNFa in vitro. The transformant was screened by vector primers and gene specific primers, and the transformant plasmid DNA was purified and sequenced. The sequence result shows that recombinant TNF (rTNF) plasmid DNA was constructed successfully. The recombinants were cultured in LB media with 100U/ml ampicillin, and were induced by IPTG, the expression efficiency of rTNFa was determined by SDS-PAGE. Time course experiment indicates that rTNFa expression was increased withthe time of IPTG induction; The expression was increased rapidly within 4 hours of induction, after that the expression was increased very slowly. The recombinant TNFa was purified under denaturing conditions with the techniques of immobilized-metal affinity chromatography (IMAC) and Ni-NTA matrices, about 0.5-1 mg of the purified rTNFa was obtained from 1 liter of bacterial culture under the denaturing condition. Finally, the purified rTNFa was refolded by buffer exchanged with phosphate buffer solution. Due to the solubility of the recombinant BS TNFa produced by the induction, the BS rTNFa could be also purified under native condition.