Molecular Cloning And Expression Analysis Of TNF,IL-8 And HSP70 Gene Of Silver Carp (Hypophthalmichthys Molitrix)

Cytokines are a group of small-molecule peptide.They mainly play fundamental roles in the development,inflammation,and function of the immune system.HSP70 is a kind of important endogenous cell-protective factor which has a variety of biological activities in immunoregulation.Silver carp(Hypophthalmichthys molitrix)is an important aquacultured fish species.Culture and some physiological factors of Silver carp are most reported,but up to now,few researches of immunity of silver carp are available.In order to study the defense mechanism of silver carp,in this paper,we have cloned and sequenced the full-length cDNA of TNF and the core cDNA of HSP70 by homology cloning approach combined with RACE method.The expression pattern of silver carp TNF in various tissues was studied by using RT-PCR.Finally,the IL-8 was overexpressed as a fusion protein in E.coli M15.The full-length TNF cDNA of silver carp was 1254 bp,and the cDNA sequence contained 394 bp 5' UTR,398 bp 3' UTR and 462 bp open reading frame(ORF) which could translate a putative peptide of 239 amino acids including two cysteines,TNF family signature,and two endotoxin-responsive sequence(TTATTTAT) of TNF-α.The obtained HSP70 core sequence was 1428 bp in length,encoding 370 amino acids residues which contained a HSP70 family signature.Phylogenetic analysis of amino acid sequences encoded by TNF of silver carp and other animals was performed by a program-MEGE4.The TNF phylogenetic tree showed that silver carp TNF was type-αTNF,and it was closest to that of common carp and more evolutionarily related to that of channel catfish of Siluriforrn,but less related to those of other two species of Pleuronectiform and Perciform.The expression analysis using RT-PCR showed that the silver carp TNF gene was constitutively expressed in the head-kidney,brain and branchia of the unstimulated fish.The IL-8 gene was directionally cloned into expression vector pQE30 to construct pQE30-IL8 recombinant.An expected 11 kD protein was tected by the SDS-PAGE analysis.