Identification And Function Of Grass Carp(Ctenopharyngodon Idellus) Interleukin 21 And Its Receptor IL-21Rα

Interleukin 21(IL-21)is produced by CD4~+T cells that are stimulated and perform various biological functions through receptors.Many studies have shown that in mammals,IL-21 affects the proliferation,survival,differentiation and function of various immune cells,and participates in the regulation of immune response homeostasis.In mammals,interleukin-21 receptor(IL-21R)is mainly expressed in spleen,thymus,other lymphoid tissues and some non-immune cells,such as intestinal epithelial cells,fibroblasts,keratinocytes and endothelial cells.Therefore,IL-21 acts on many cell types.According to the cell types of receptor distribution,IL-21 plays an important role in the regulation of immune response.With the change of global climate,diseases occur frequently in aquaculture industry in recent years,which directly cause huge economic losses to aquaculture producers.For the increasing production demand of Ctenopharyngodon idellus,we need to further study and analyze the immune response of Ctenopharyngodon idellus under the condition of pathogen infection.In order to further understand the expression and function of grass carp IL-21 in pathogen stimulation.In this study,the homologues of IL-21 and IL-21R,Ci IL-21(Ctenopharyngodon idellus interleukin-21)and Ci IL-21Rα(Ctenopharyngodon idellus interleukin-21 receptorα-chain),were identified by PCR and bioinformatics analysis.The expression of Ci IL-21 and Ci IL-21Rαin kidney,spleen,gill and intestine were analyzed by intraperitoneal injection of Flavobacterium clourane(FC)and grass carp GCRV-II virus,and real-time quantitative PCR.The expression of IL-1,IL-8,IL-10,IL-22,Ci IL-21Rα,IFN-γin the primary cells isolated from the kidney of grass carp was detected.In order to further understand the subcellular localization of Ci IL-21 in tissues stimulated by pathogens,r Ci IL-21 was used to prepare monoclonal antibodies of mice,and then through frozen section technology and laser confocal microscope,after intraperitoneal injection of grass carp with the same volume of FC and PBS,the middle kidney and hindgut of grass carp were taken for the subcellular localization of Ci IL-21.The main results are as follows:1.The sequence coding for aminoacids in protein(Ci IL-21)region was 450 bp in length,with 6 exons and 5 introns.Its structure was consistent with that of zebrafish IL-21.It encodes 149 amino acids,19 amino acid size signal peptides,4 conserved cysteine and 4 alpha helix domains which are consistent with the characteristics of type I cytokines.By constructing phylogenetic trees,we identified the homologous relationship between Ci IL-21 and known members of IL-2 family.It was found that zebrafish and grass carp IL-21 were clustered into one twig with a bootstrap value of 100%,which had high homology.2.Obtain Ci IL-21Rαthe total length of CDS region is 1593 bp,including 7 exons and 6 introns.The structure of CDS region is consistent with that of zebrafish IL-21R.1.The total length of CDS region is 530 amino acids,including 34 amino acid size signal peptides,213 amino acids in extracellular region,typical type I cytokine receptor domain,two conserved cysteine and one conserved WS-X-WS amino acid motif in the front of extracellular region.By constructing phylogenetic trees,we identified the homologous relationship between Ci IL-21Rαand IL-2Rα,IL-15Rα,IL-21Rα.It was found that the IL-21Rαof zebrafish and grass carp were clustered into one twig with a bootstrap value of 100%,which had high homology.3.Tissue expression and pathogenic infection analysis of Ci IL-21 and Ci IL-21Rα.The expression of Ci IL-21 in gill,intestine,body kidney,head kidney,spleen,skin,thymus and liver were obtained by real-time fluorescence quantitative PCR.The results showed that Ci IL-21 was the highest in gill,intestine and the lowest in skin;Ci IL-21Rαwas the highest in spleen and gill and the lowest in liver.Four tissues of gill,intestine,kidney and spleen were collected at one day,two days and three days respectively in the infection experiment of FC.It was found that Ci IL-21 and Ci IL-21Rαwere significantly up-regulated.In GCRV-Ⅱinfection experiment,four tissues of gill,intestine,kidney and spleen were collected at 1 d,3 d,7 d and 14 d respectively to study the expression of Ci IL-21 and Ci IL-21Rα.The results showed that Ci IL-21 and Ci IL-21Rαdecreased first,then increased,and all of them showed significant changes.4.Obtained r Ci IL-21 and r Ci IL-21Rαrecombinant proteins.Order to evaluate the biological activity of r Ci IL-21,the recombinant protein was purified by AKATA system.Then,the primary renal leukocytes isolated from the kidney of grass carp were stimulated with r Ci IL-21 protein of 5 ng/ml,50 ng/ml and 500 ng/ml,respectively.When the stimulation concentration was 50 ng/ml,IL-10,IL-22 and IFN-γwere all significantly up-regulated,but no significant changes were detected in other cytokines.5.The specific monoclonal antibody of Ci IL-21 was obtained.Ci IL-21 protein was obtained from prokaryotic expression system,eukaryotic expression system and grass carp.Western blot was used to verify the specificity of Ci IL-21 monoclonal antibody,and the monoclonal antibody with better specificity was screened.6.Immunofluorescence localization of Ci IL-21 in kidney,intestine and gill of grass carp was studied by frozen section and laser confocal technique.After FC infection for24 hours,the distribution of Ci IL-21 in kidney and intestine increased,but not in gill.The above results showed that Ci IL-21 and Ci IL-21Rαplayed a role in the immune response of grass carp against the invasion of pathogens.Ci IL-21 can play an immunomodulatory role by up regulating T helper cytokines,IFN-γ(Th1),IL-10(Th2)and IL-22(Th17).