Cloning,Sequence Characterization Analysis of the Tumor Necrosis Factor alpha-1(TNFα1) Full-length cDNA from Carp

The cDNA library of peripheral blood leucocytes which were separated from carp(Cyprinus carpio L.) and stimulated with mitogen was screened by a probe labelled with DIG.The tumor necrosis factor alpha(TNFα1) EST sequence was picked out from the cDNA library of peripheral blood leucocytes that was constructed.After two rounds of screening from 0.9×104 reeombinate phages,the positive clone was obtained.Sequence analysis indicated that it had a 128 bp 5′-UTR and a 423 bp 3′-UTR which included the instable motifs of ATTTA.The open reading frame(ORF)of this gene was 768 bp which putatively coded 255 amino acids with the MW of 28.1 ku,and PI of 8.20.Its homology with TNF from GenBank was up to 94%.Analysis of protein sequence and structure,within the predicted protein sequence we identified a TNF family signature,a predicted transmembrane domain and a putative cleavage site to produce the mature peptide.