Discovery And Functional Characterization Of A Novel Lineage Specific Interleukin In Bony Fish

Cytokines,including interleukin and interferon,have a variety of functions that regulate both innate and adaptive immunity.Numerous cytokines play a role in a variety of secretory ways such as paracrine and autocrine in the organism,forming an extremely complex network of cytokine regulation and participating in various important physiological functions of the organism.As the lowest vertebrates,fish has both innate immunity and adaptive immunity,and is an extremely important research object in immune evolution research.In this paper,a model animal zebrafish was used as a research object,and a novel cytokine specific to fish was obtained by bioinformatics analysis.The cytokine was similar in gene structure to zebrafish interleukin or interferon,and Zebrafish interleukin 22,interleukin 26,gamma interferon I and its receptor are located adjacent to chromosome 4 of zebrafish,so we temporarily named it FIL(Fish-specific Interleukin/Interferon like molecule).To further explore the function of this novel cytokine,we first obtained the FIL gene length of 1243 bp by RACE technology,including 639 bp of CDS region,and then detected the expression of FIL in healthy zebrafish tissues by real-time PCR,the results showed that FIL was The expression level of mucosa in the hindgut,sputum,skin and the like of healthy zebrafish is high.Then we inoculated the zebrafish by intraperitoneal injection of POLY I:C,Edwards ellada(Et)and Hunchun viremia virus(SVCV),and detected by fluorescence quantitative PCR.The zebrafish is rapidly expressed in the spleen after infection with the virus and its analogs,and is expressed in the intestines at a lower level.After bacterial infection,FIL plays an important role in mucosal tissue.To further explore the biological function of FIL,we constructed the prokaryotic expression plasmid and eukaryotic expression plasmid of FIL,and successfully purified to obtain prokaryotic and eukaryotic recombinant proteins.At the same time,we prepared a polyclonal antibody against zebrafish FIL.Western blot analysis showed that the polyclonal antibody specifically recognizes FIL recombinant protein.Subsequently,we purified the prokaryotic expressed protein to stimulate the zebrafish ZF4 cell line according to different gradients to understand the biological activity and biological function of the FIL protein at the cellular level.The transcribed group was recorded after stimulation,and the results showed that FIL plays a role in zebrafish antiviral immunity and inflammatory response.We selected the corresponding target gene for real-time PCR to verify the results obtained by the transcriptome.Next,we hope to further identify its taxonomic status by analyzing the crystal structure of FIL molecules.We crystallized the purified high-purity prokaryotic protein,obtained high-quality protein crystals,and optically diffracted the crystal,collected structural data of the crystal,but compared with other protein sequences in the protein structure database.We found that although the zebrafish FIL gene is highly similar to interleukin and interferon in amino acid sequence,it has low homology with protein structure prediction.Therefore,we need to obtain the seleno derivative of FIL protein and obtain its crystal,further diffracting.The conditional exploration of selenoprotein crystallization is reflected in this paper,and the crystal diffraction data is to be obtained.Through a series of work in this experiment,we have further identified the biochemical function,gene structure and protein conformation of the zebrafish cytokine FIL,which lays a foundation for the follow-up study of this cytokine.The S100 family proteins are a group of small acidic polypeptides and have diverse functions in regulating many aspects of physiological processes.They are structurally conserved,possessing two EF-hands which are central for calcium-mediated functions.The S100 family proteins are known to be involved in host defence,such as regulating calcium hemostasis and cell migration.Although the S100 family genes have been described in teleost fish but their roles in immune defence are poorly investigated.Thirteen S100 cDNA sequences were determined in zebrafish and their genomic organizations confirmed.Re-analyzing the gene synteny of the S100 loci revealed that two major S100 loci were present in Chr16 and Chr19,comprising 13 tandemly-linked S100 genes and were believed to share a common origin with the S100 locus in human Chr1.The two S100 zebrafish loci is postulated to have evolved from a single locus during the teleost specific whole genome duplication event.It seemed that the homologues of human S100 G and S100 P have been lost in zebrafish.Constitutive expression was examined in systemic(head kidney and spleen)and mucosal(gills and gut)immune organs,indicating that the S100 genes are relatively highly expressed in mucosal tissues.Intraperitoneal injection of poly(I:C)resulted in up-regulation of most S100 genes in the gut,notably,marked induction of S100V2 and S100 Z expression,and in the spleen at 48 h post injection.In zebrafish challenged with spring viremia of carp virus(SVCV),expression of most S100 genes was increased in the spleen between day 1 and day 7 post infection,with consistent induction of S100A10 a,S100A10b,S100A10ICN1,S100A10 U,S100V1 and S100 Z observed.Intraperitoneal injection of E.tarda down-regulated expression of S100 genes in the gut but increased S100 genes in the spleen at 72 h post injection.Fish S100 genes share a common origin with mammalian counterparts.Zebrafish S100 genes are differentially modulated by bacterial and viral pathogens and are involved in immune defence in fish.