| Objective Interleukin8(IL-8), or CXCL8, is a CXC chemokine that is known tomediate inflammatory response in mammals. Until now, variety of fish IL-8genes hasbeen cloned, and their functions have been studied priliminarily. Grass carp(Ctenopharyngodon idella) is a major freshwater fish species in China, Even being thethird largest breeding species in the world. Grass carp is vulnerable to infections andthen burst into inflammation even death, that seriously obstructs the development ofgrass carp aquaculture. This thesis intends to clone the complete gene of IL-8in grasscarp (CiIL-8), and preliminarily identify its biological function.Methods Firstly, We cloned full length cDNA sequence of IL-8from the grass carpby making use of Rapid Application of cDNA Ends (RACE), and then subcloned it intothe prokaryotic expression plasmid pET32a+to obtain IL-8fusion protein. Secondly, wetested mRNA levels of IL-8in blood and twelve other tissues of healthy grass carp bymeans of Real-time PCR, and we also checked the fold-changes of mRNA levels ofIL-8in brain, heart, intestinal, kidney, spleen, head kidney, gills, thymus, skin, the tailfin and liver of infected grass carp after intraperitoneal injection of Aeromonashydrophilia for4hours,1day,3days and7days respectively. Finally, we observedthe pathological morphology changes of intestinal in the infected grass carp asmentioned above by utilizing Scanning Electron Microscopy (SEM) and ParaffinSection.Results The length of Grass carp IL-8cDNA sequence is609bp, including5’ UTR96bp,3’ UTR216bp and ORF297bp which encodes98amino acids, Sharing45%identity with the human IL-8. The predicted third structure of CiIL-8structure is similar to human IL-8. CiIL-8genomic DNA including four exons, and its arragement of exonsand introns is similar to IL-8in other species; CiIL-8fusion protein is successfullyobtained, being detected with the correct molecular weight. The Real-time PCR resultsof blood and twelve tissues of health grass carp showed that IL-8is transcribed inervery tissue. In comparison, the highest level showed in the liver, the tail fin and skin issecond only to the liver, immune related organs such as trunk kidney, spleen, intestinal,head kidney, gill, thymus also showed ralatively high levels. After intraperitonealinjection of Aeromonas hydrophilia, IL-8mRNA fold changes were examined in eleventissues. There was no change in the brain and the fins, the other nine tissues (heart,intestinal, trunk kidney, spleen, head kidney, gill, thymus, skin and liver) respectivelyreached a peak in4h or1d. Among them, the immune related tissues showed thehighest increase, and they all recoverd at day3. After artificially infected grass carp byAeromonas hydrophilia, the pathological morphology changes in intestinal described asbelow: endothelial cells showed vacuolar degeneration in4h, At day1the reticulartissue of submucosa showed obvious symptom such as congestion, cell swelling, withlamina propria lymphocytic infiltration,3days after injection, lymphocytes infiltrated inreticular tissue of submucosa. At day7after injection the intestinal tissue was revovered.Ultrastructure of intestinal villus showed the obvious breakage at day3, and seemednormal at day7.Conclusion In the current studies, we cloned and characterized a homologous geneof IL-8, which is an essential and well known inflammatory cytokine. This grass carpIL-8gene is constitutively expressed in multiple tissues of grass carp at various levels;Expression of IL-8could be induced by pathogenic bacteria infection rapidly (within4hour) and reach its peak at1day in immune related organs, then IL-8levels graduallydroped down to its basal level after3days. While in intestinal tissue, IL-8levelsreached its peaks3days after infection, and returned to its basal level after about7days.In conclusion, we identified IL-8homologous gene in grass carp for the first time, andcharacterized the essential functional role of grass carp IL-8in immune response andimmune regulation. |
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