Abstract: There are two distinct Tumor necrosis factor alpha (TNFα 1 and TNFa2)genes in the genome of rainbow trout ( Oncorhynchus mykiss ). The two TNF α genes share 94% and 92% identity in nucleotide and amino acid, respectively. This phenomena is different from that in mammals which containing only one copy of TNFα gene in the genome. In order to investigate the biological functions of the two TNFα homologues in immune response, these two TNFα genes were recombined and expressed in prokaryotes to produce recombinant proteins in vitro. Both of the two TNFα mature peptide coding fragments were amplified from plasmid pT1 and pT2 which contained TNFα gene, using gene specific primers covered BamHI and HindIII sites. The PCR products and pQE30 expression vector were digested by BamHI and HindIII, respectively, and then purified by gel purification. The purified products were pooled together and ligated by T4 ligase at 14℃ overnight. Subsequently, the ligation products were transformed into E. coli strain JM109 and cultured at 37℃ overnight. The transformants were screened by vector primers and gene specific primers, and then identified by DNA sequencing. The recombinants TNF (rTNFs)were cultured and induced to express the recombinant proteins, then determined by SDS PAGE. Expression results showed that both of the two rTNFs were expressed at high level and formed insoluble aggregates (inclusion bodies) in the cytoplasm of E. coli with or without inducement of IPTG, the yield of expression rTNFs was about 25%-30 % of gross bacterial protein. Due to the inclusion body, the recombinant proteins could only be purified under denature condition using 8M urea to solubilize the inclusion bodies, and then purified with the techniques of immobilized metal affinity chromatography (IMAC) and Ni NTA matrices. The 6 His tag with the recombinant proteins can attach the Ni NTA matrices to separate the other proteins in high pH condition, and can be eluted in low pH condition. About 0.5-1mg/L purified recombinant proteins could be obtained from the cultured bacteria with this approach.