Expression And Functional Analysis Of Cytokines IL-22,IL-26 And EDA In Grass Carp (Ctenopharyngodon Idellus)

According to the 2020 edition of China Fisheries Statistical Yearbook published by China Agriculture Press,the total output of grass carp in China in 2019 was 5.533 million tons,and the total output of freshwater aquaculture in China was 30.137 million tons,the total output of grass carp cultivation accounted for about 18.4 percent of the total output of freshwater cultivation in China.Therefore,grass carp is one of the most important objects of aquaculture production in China.However,with the development of grass carp breeding industry,diseases occur frequently,such as the third disease of grass carp,grass carp hemorrhagic disease and various new diseases,which seriously hinder the healthy and sustainable development of grass carp breeding industry.Therefore,it is an urgent problem to analyze the immune molecular mechanism of grass carp and seek new methods for disease prevention and control.Many cytokines in vertebrates are closely related to the immune system.Interleukin and tumor necrosis factor family are important representatives of them,and play an important role in various immune responses.Studies have shown that interleukin can participate in the regulation of the body's inflammatory response and the polarization and migration of macrophages,enhance the phagocytic activity of phagocytes,participate in humoral and cellular immune response,change antibody types,and promote the maturation and differentiation of T lymphocytes.Tumor necrosis factor(TNF)plays a variety of roles in immune inflammation,proliferation,differentiation and hematopoiesis.At present,it has been reported that a variety of interleukin can participate in the immune response in vertebrates,among which interleukin-22 and IL-26 are key inflammatory cytokines,but the specific biological functions in grass carp have not been fully understood.EDA gene is involved in the formation of scales on the skin,and the skin has a strong resistance to a variety of pathogenic organisms.Whether EDA is also involved in it and whether it is also involved in the body's inflammatory response has not been reported.1 Expression and functional identification of interleukin-22 in grass carpInterleukin(IL)-22 plays an important role in the regulation of inflammatory clearance of infectious pathogens.IL-22 homologues have been found in fish,but their functions and sources of IL-22 production have not been fully elucidated.In this study,a homolog of IL-22 was identified in grass carp and its biological activity was studied.IL-22 was constitutively expressed in tissues of grass carp,with the highest expression in gills and hindgut.The expression of IL-22 was up-regulated in the spleen after infection with Flavobacterium columnaris,Aeromonas hydrophila and grass carp reovirus,and in the primary head kidney and spleen leukocytes stimulated by LPS,IL-2,IL-21 and IL-34.On the contrary,it is down regulated by Th2 cytokines such as IL-4/13B and IL-10.The expression of inflammatory cytokines,STAT3,SOCS3 and antimicrobial peptides in primary head kidney leukocytes and CIK cells were enhanced by recombinant IL-22.In addition,IL-22 positive cells were induced in hindgut and head kidney 24 hours after infection with F.columnis by using monoclonal antibody(GC3-22)against recombinant protein under confocal microscope.It was also found that IL-22 could be secreted by CD4~+T cells under inflammatory conditions,and IL-22 could reduce the apoptosis induced by CCCP on CIK cells.The study also analyzed the structure of grass carp IL-22protein and found that it has a typical IL-10 family cytokine structure.Our data suggest that IL-22 plays an important role in regulating mucosal and systemic immunity against bacterial and viral infections.2 Expression of interleukin-22 receptor 1 in grass carpThe functional receptor protein of IL-22 is a complex composed of two subunits,IL-22RA1 and IL-10R2,which is the only on-membrane receptor of IL-22,among which IL-22RA1 is a specific receptor for IL-22,while IL-10R2 is a common receptor for most members of the IL-10 family.IL-22 in higher vertebrates transmits signals into the cell through the dimer of IL-22RA1-IL-10R2 protein,and this is the only way that IL-22transmits signals,which has been well established in mammals.However,there is little information about the molecular characterization and role of the IL-22 receptor in osteoclast in inflammatory response induced by pathogenic microorganism infection.IL-22RA1 homologue was identified from grass carp according to the chromosome location and gene homology.It was predicted that IL-22RA1 homologue has signal peptide and transmembrane region,which is a typical membrane structural protein.Analysis of tissue distribution of grass carp IL-22RA1 showed that its expression level was the highest in hindgut and gill.Under the stimulation of bacteria and other pathogenic microorganisms,the expression of IL-22RA1 was significantly up-regulated.Various inflammatory cytokines can also induce the expression of IL-22RA1 in different cells.The recombinant extracellular domain protein of His-rCiIL-22RA1 was prepared in prokaryotic expression system,which can provide support for further study on the biological function of grass carp IL-22.3 Expression analysis of interleukin-22 extracellular binding protein(IL-22BP)in grass carpIL-22 binding protein(IL-22bp)is a natural soluble single chain as a functional inhibitory receptor of IL-22.Il-22BP can compete with IL-22 receptor protein complex,directly bind to IL-22,prevent the interaction between cytokine and cell surface receptor complex,and neutralize its activity.Based on the homologous gene sequence,the partial series of grass carp IL-22BP was found in the CDS database of grass carp genome by Local Blast,and then the primer clone was designed to identify the grass carp IL-22BP.rCiIL-22BP is a typical exocrine protein,because it contains a signal peptide of 24 aa without a transmembrane region.Through q PCR analysis,the results showed that the expression level of IL-22BP was the highest in the skin,and it was down regulated in the early stage of pathogenic microorganism infection.Anti-inflammatory cytokines and LPS could up regulate the expression of IL-22BP in grass carp.The recombinant protein of grass carp IL-22BP was prepared,and the monoclonal antibody was also prepared.The study also found that grass carp IL-22BP could be secreted by CD4~+T cells under inflammatory conditions.Only part of grass carp IL-22BP protein was analyzed structure.This study can provide support for further exploring the role of IL-22-IL-BP in the inflammatory response of teleost.4 Interaction between IL-22 and its receptor in grass carpThe interaction of IL-22 and its receptors(IL-22R and IL-22BP)plays an important role in mammalian inflammatory response.In lower vertebrates,especially in teleost fish,there are few studies on the interaction between IL-22 and its receptor.The purpose of this study is to study the interaction between IL-22 and its receptor at cellular and protein levels by using immunoprecipitation,immunofluorescence,q PCR and Western blot,and to further explore the biological function of IL-22.Based on the above studies,using immunoprecipitation reaction,it was proved that grass carp IL-22RA1 and IL-22BP were the receptors of IL-22 in vitro.q PCR analysis showed that IL-22BP could inhibit the biological activity of IL-22 protein in vitro.Western blot analysis confirmed that grass carp IL-22 could activate STAT3 phosphorylation through IL-22RA1-10R2 complex receptor,thus mediating biological function.Immunofluorescence technology showed that grass carp IL-22 and IL-22BP could be secreted by CD4~+T cells under inflammatory conditions.This study further confirmed that IL-22 mediates inflammatory response through interaction with receptors in teleost fish.5 Expression and functional identification of interleukin-26 in grass carpIL-26 is an important member of the IL-10 family and a proinflammatory cytokine.It has been identified in humans,amphibians and teleost fish,but not in rodents.Although IL-26 has been identified in grass carp and other teleost fishes,and its inflammation related functions have been studied,the role of IL-26 in the regulation of inflammation in lower vertebrates,including teleost fishes,is not very clear.q PCR analysis showed that the expression of IL-26 in grass carp skin and head kidney was the highest;the expression of IL-26 was significantly up-regulated by pathogenic microorganisms,especially bacteria.LPS,PHA and proinflammatory cytokines could up regulate the expression of IL-26 in different cells.Grass carp IL-26 could up regulate inflammatory cytokines,STAT3 and some antimicrobial peptides.In this study,we also prepared prokaryotic recombinant protein of grass carp IL-26,and prepared monoclonal antibody of IL-26.Screened the crystal growth conditions of grass carp IL-26 protein.We also found that grass carp IL-26 has certain biological function of inducing apoptosis.This study further enriched the theoretical knowledge of IL-26 in teleost.6 Cloning and expression analysis of ectodysplasin-a gene and its receptor in grass carpEDA is a membrane protein,belonging to the TNF family,and its receptor is EDAR.EDA and its receptor EDAR have been considered to be related to the growth and development of animal hair,teeth and bones.Recently,EDA and its receptor EDAR homologues have been found in fish,and their relationship with fish scale growth and evolution have been studied.Based on gene homology,the full length of EDA and EDAR genes of grass carp were cloned and identified,and their protein structures were predicted by bioinformatics analysis.It was found that grass carp EDA protein is a typical non secretory protein without signal peptide and transmembrane domain,while EDAR is a typical membrane protein with transmembrane domain and signal peptide.The results of q PCR showed that the expression level of EDA in skin was highest,while the expression of EDAR was the highest in spleen.Pathogenic microorganisms,especially bacteria,can significantly induce the expression of EDA and EDAR in grass carp,especially in skin.LPS,PHA and inflammatory cytokines up-regulated the expression of EDA and EDAR in different cells.This study suggests that EDA and its receptor EDAR are involved in the inflammatory response of teleost,but further study is needed.