| Spotted sea bass(Lateolabrax maculatus),belonging to perciforms and servanidae.Interleukin 17(IL-17)family is a class of cytokines,which are secreted proteins of small molecules and play an important role in both innate and adaptive immunity through specific binding with receptors to regulate a series of intracellular reactions.In this study,four ligands(IL-17A/F1,IL-17 B,IL-17 C,IL-17D)and one receptor(IL-17RB)of the IL-17 family of spotted sea bass were identified,and their molecular structures were analyzed by bioinformatics method.By real-time quantitative PCR(q RT-PCR)method to analysis the five gene expression in the healthy spotted sea bass organizations.After spotted sea bass infecting Edwardsiella tarda,five gene expression in immune related organization(gill,head kidney and spleen)were analysed.The effects of LPS/ Poly(I:C)stimulation on the expression of five genes were analyzed.Prokaryotic expression vectors were constructed to obtain IL-17 B and IL-17 RB recombinant proteins of spotted sea bass.The results are as follows:(1)Four IL-17 ligands and one IL-17 receptor c DNA of spotted sea bass were obtained by cloning.Sequence analysis shows that IL-17 ligands except IL-17 C have predicted signal peptides.Each IL-17 ligand has four conserved cysteines that form disulfide bonds.IL-17 receptor(IL-17RB)also has a predicted signal peptide.IL-17 RB is a single transmembrane protein.(2)Through q RT-PCR analysis,it is found that each gene of spotted sea bass has different expression levels in different tissues.We analysis the expression of IL-17 genes in gill,head kidney,intestine,spleen,liver,brain,skin and muscle.The expression of genes were detected in all tissues.The expression of IL-17A/F1 gene in healthy spotted sea bass was the highest in gill and the lowest in muscle.The expression of IL-17 B gene in healthy spotted sea bass was the highest in gill and the lowest in liver.The expression of IL-17 C gene in healthy spotted sea bass was the highest in muscle and the lowest in head kidney.The expression of IL-17 D gene in healthy spotted sea bass was the highest in brain and the lowest in muscle.The expression of IL-17 RB gene in healthy spotted sea bass was the highest in gill and the lowest in intestine.Gill is the first line of defense against pathogen invasion and an important mucosal immune organ of fish.The high expression of most IL-17 genes in gill indicates that they play an important role in mucosal immune regulation.The expression levels of IL-17 B and IL-17 RB gene were similar,which might be related to their specific binding.(3)The results showed that the IL-17A/F1 gene in gill was significantly downregulated at 12 h after injection of LPS,Poly(I:C)and Edwardsiella tarda,respectively.IL-17A/F1 gene expression levels were different in each specie,and the specific role of IL-17A/F1 was difficult to distinguish.At 6 h after injection of LPS,Poly(I:C)and Edwardsiella tarda,IL-17 B gene was significantly up-regulated in head kidney,indicating that IL-17 B respond rapidly to pathogen invasion.At 6 h after LPS injection,IL-17 C expression in the head kidney was upregulated 6 times,suggesting that IL-17 C may play a unique role in the initial stage of inflammation.IL-17 D was significantly upregulated in the head kidney at 48 h after injection of LPS,Poly(I:C)and Edwardsiella tarda,confirming the involvement of IL-17 D in the inflammatory response.The expression of the IL-17 RB gene in head kidney is significantly upregulated at 6 h after Edwardsiella tarda injection,which was consistent with IL-17 B.(4)Recombinant plasmids of IL-17 B and IL-17 RB were constructed respectively,and the recombinant proteins of IL-17 B and IL-17 RB were obtained after a large amount of induction,and the recombinant proteins were purified to lay the foundation for the next functional experiment.SDS-PAGE showed that the size of the recombinant proteins were consistent with the predicted results. |
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