Cloning,Identification,and Immune Response Of Trachinotus Ovatus IL-17A

The intereukin17(IL-17)subfamily is an important class of pro-inflammatory cytokines.In this study,we took Trachinotus ovatus as the research object,cloned the gene IL-17A(TroIL-17A),and analyzed its sequence characteristics,antibactiarial function and protein functions.The results are as follows:(1)The TroIL-17 A open reading frame contains a base of 426 bp and encodes 142 amino acids,including a signal peptide of 23 amino acids.The amino acid sequence encoded TroIL-17 A has 19.01%-76.43% similarity to mammal such as human,mice,et al and other bonefish IL-17 A.Among them,the IL-17 A sequence homology between TroIL-17 A and Miichthys miiuy IL-17 A is the highest at 76.43%,and the lowest is 33.33%with Ctenopharyngodon idella among the teleost.And compared with the TroIL-17 A sequence,IL-17 A of human has only 19.01% homology.Indicating that the conservation of IL-17 A between species is not very high.Phylogenetic Tree results indicate that TroIL-17 A has the closest reletive with catfish and European tongue bass which belong to Perciformes.(2)TroIL-17 A is expressed in 11 organs which conclude liver,spleen,head and kidney,skin,muscle,stomach,intestine,gill,brain,heart,and blood.The highest expression was in the immune organs liver,head kidney and spleen.When healthy fish were infected by Vibrio harveyi,Edwards lardii,Streptococcus agalactiae and neuronecrosis virus,the expression of TroIL-17 A in the liver and head kidney was significantly increased.Therefore,the immune gene TroIL-17 A is important immune factors in the body immune regulation.(3)The TroIL-17 A overexpression group can significantly inhibit the infection of fish by V.harveyi.After the expression of TroIL-17 A is disturbed,the amount of bacterial infection increases significantly.This shows that over-expression of TroIL-17 A can enhance the body’s immunity and significantly increase the antibacterial immunity of T.ovatus.However,after interfering with the expression of TroIL-17 A,the body’s immunity decreases,making T.ovatus more susceptible to infection by pathogens such as bacteria.(4)Through subcellular localization experiments,it shows TroIL-17 A is expressed in the whole-cell.In this study,we used prokaryotic expression system pET-32a(+)/E.coli BL21(DE3)to induce the expression of the recombinant protein rTroIL-17 A.And we got pure protein rTroIL-17 A by nickel ion column affinity chromatography purification.We also prepared a polyclonal antibody.Further research on the function of the protein showed that the protein can induce the migration of peripheral blood leukocytes(PBL)and proliferation of head kidney lymphocytes(HKL).At the same time,the recombinant protein rTroIL-17 A has positive effects to the gene expression of IL-6,IL-8,TNF-α,IL-1β.Therefore,TroIL-17 A plays an important role in chemotaxis of PBL,proliferation of HKL,and regulation of other cytokines.In summary,this study demonstrates that TroIL-17 A has the functions of chemotactic peripheral blood leukocytes,proliferating lymphocytes,and regulating other cytokines.These results This reseach has laid the foundation for the research on the immune factors of teleost fish,provide a new way for disease prevention and control of T.ovatus.