| As the world′aquaculture one of the most developed countries, in national economy fresh water industry occupy an important position, and along with the improvement of living stadards, aquaculture also have much room for development and the construction of the aquatic animals diseases, particularly the prevention and control of infectious diseases has put forward higher requirements. However, as the most effective prevention of infectious diseases immunization means, in the use of aquatic animal disease control, there are still many restrictions, with the current depth study of fish immune lower, particularly at the molecular level research is very much related to the deficiencies. Therefore, strengthening the fish molecular immunology basic research is necessary. IL-8 as an important cytokine, and its biological activity mainly in the peripheral blood neutrophils and T lymphocytes chemokines, can lead to intracellular free Ca2+ concentration of particles rapid and transient increase, and deformation induced cell response degranulation responses to the outbreak of respiratory and lysosomal enzyme release, the cells can be activated. On neutrophil chemotactic cytokines, the role of IL-8 and vascular endothelial cells interaction makes IL-8 become an important pro-inflammatory cytokines in the body of the antibacterial immune process plays a key role. At present carp IL-8 the genome sequence and genome structure, as well as its mode of transcription was induced by the expression of regulatory mechanism is not yet clear, this is not only concerned about the content of this study is the significance of the study.This study used the DD-RTPCR obtained differential display fragments A26, the digoxin labeled as a probe of nucleic acid hybridization screening mitogen-stimulated carp peripheral blood leukocytes cDNA library from 0.8×104 recombinant phage. After two rounds of screening positive cloning, sequencing analysis confirmed that the IL-8 for the full-length cDNA that the mitogen stimulation, in the carp peripheral blood leukocytes was IL-8 gene expression. After obtaining the carp IL-8 cDNA cloning, in its bilateral non-translated region, primers were designed to extract the carp spleen genomic DNA as template for PCR amplification, will be amplified product recovery and purification, to connect to pMD18- T vector and transformed into competent E. coli DH5αcell, and insert fragments were sequenced, access to the carp interleukin-8 full-length genomic DNA sequence. In addition, the study also extracted under different conditions by LPS,PHA and ConA stimulation of total RNA, according to the carp IL-8 full-length cDNA sequences andβ-actin sequence, primers were designed using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method of isolation and analysis in the normal leukocytes and mitogen-stimulated different times in the gene transcription discrepancy. The results showed that: the carp IL-8 full-length cDNA clone are 609 bp, and it was registered for DQ453125 in GenBenk, containing 5-untranslated region and 3-untranslated region and 3 ? poly (A) tail and signal (AATAAA) and poly (A) tail, a middle-size 294 bp encoding 98 amino acids complete open reading frame. Amino acid sequence comparison found that people, pigs and other mammals conservative sequence of about 70 percent, while fish is relatively low, compared to only about mammals and 30 percent conservative. Also found that people, monkeys, pigs, dogs, cats, cows, chickens, black cod, the seals in the CXC have ELR front-sequence motifs, and so far the fish found in the IL-8 are ELR sequence incomplete, some are LR (such as salmon), and some only E (such as flounder), or R (like carp), and some are not. We can speculate: bony fish IL-8 did not complete ELR sequence, in the subsequent evolution of the ELR-produced sequence. At the same distance from the evolution of mammals and fish under comparison, the molecular evolution of fish faster than mammals. Full-length genomic DNA of 943 bp was registered for EU011243 in GenBank, the gene sequence analysis showed that the evolution of conservative, and like all other species contains 4 exons and 3 introns, and the people, pigs, dogs, rainbow trout gene structure comparative analysis showed that the IL-8 gene structure and function of the regional continuity of the conservative way to the evolution of fish each exon base in the relatively few mammals, and should be the base of exon basically the same, and the exon-intron splice site GT-AG to comply with the rules, only a portion of exon larger changes, we can see that the gene in fish faster between the evolution of the molecular and genetic evolution mainly concentrated in the intron of the changes; transcription analysis showed that: carp peripheral blood leukocytes stimulated with LPS and PHA for 4 h than normal for 4 h of IL-8 expression increased (P <0.01), and stimulated with LPS and PHA for 12 h than normal for 12 h of IL-8 expression declined (P <0.01). ConA stimulated for 4 h than normal for 4 h of IL-8 expression increased(P> 0.05), 24 h ConA stimulated than normal for 24 h of IL-8 expression of a diameter slightly larger (P> 0.05). The stimulated white blood cells and the normal IL-8 expression trends into peak shape map. The difference is that the stimulation of IL-8 expression of the peak reached in advance, suggesting that IL-8-induced transcription of the different sensitive varying degrees, and that IL-8 may be early in the cells stimulated immune to play the role.
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