Molecular Cloning And Analysis Of IL-8,TNF,CXCR1,CXCR2 And HSP70 Gene Of Silver Carp (Hypophthalmichthys Molitrix)

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, we used RT-PCR and RACE-PCR to obtain the cDNA sequences of IL-8, TNF, CXCR1, CXCR2 and HSP70 of this species, and deduced their corresponding amino acid sequences.The full-length IL-8 cDNA of silver carp was 679bp, and included an open reading frame(ORF) of 297bp for encoding a peptide of 98 amino acid which contained a CXC family signature and the ELR motif. The obtained TNF core sequence was 860bp in length, composed of an ORF encoding 153 amino acids residues which contained a TNF family signature, a TNF2 family profile, two conservative residues Cys and two endotoxin-responsive sequences(TrATTTAT) of TNF-a. The length of the obtained CXCR1 core sequence was 646bp, encoding 55 amino acids. The length of the obtained CXCR2 core sequence was 481bp, encoding 150 amino acids. The length of the obtained HSP70 core sequence was 988bp, encoding 329 amino acids which contained a HSP70 family signature.A phylogenetic analysis of amino acid sequences of five obtained genes of silver carp and other animals was performed by a program-MEGA4. The phylogenetic tree based on IL-8 showed that silver carp IL-8 belonged to the type-CXCa Chemokine family, and it was closest to common carp, then to rainbow trout of Slmoniform, but less related to other three species of Pleuronectiform, Tetrodontiform, and Gadiform. The TNF phylogenetic tree showed that silver carp TNF was type-a TNF, and it was closest to common carp and more evolutionarily related to channel catfish of Siluriform, but less related to other two species of Pleuronectiform and Perciform. The HSP70 phylogenetic tree showed that silver carp HSP70 was more close to Cypriniform species(common carp, goldfish, zebra fish) than other three species of Tetrodontiform, Pleuronectiform and Perciform.RT-PCR was used to detect the constitutive expression of IL-8 in silver carp muscle, bowel, liver, pancreas, brain, head kidney and gill. The results revealed that stronger products in the pancreas, liver and head kidney were detected, but the constitutive expression of IL-8 in brain and muscle were not detected.