Cloning And Functional Identification Of Grass Carp Interleukin 34

Interleukin 34(IL-34)is a new type of interleukin which was first reported in 2008.As the second functional ligand of macrophage colony stimulating factor receptor(M-CSFR),it has many biological functions such as promoting proliferation and differentiation of mononuclear-macrophage system,inducing the production and increase of various cytokines and chemokines,promoting inflammation,and promoting osteoclast formation.Numerous studies have shown that IL-34 plays a very important role in immunoregulation in mammals,but the function of IL-34 in teleost fish is rarely studied,especially the research on grass carp IL-34 has not been reported in the literature.This article takes grass carp as the research object:1.The IL-34 gene was cloned and identified,and its full-length cDNA was 1792 bp.The coding region sequence(CDS)consisted of 633 nucleotides,containing 6 exons and 5 introns,encoding 210 amino acids,the first 24 amino acids are predicted signal peptides,and the amino acid sequence homology with zebrafish in teleost fish is as high as 76%.Phylogenetic analysis showed that grass carp IL-34 is highly conserved in evolution.2.Quantitative real-time PCR(qPCR)was carried out for the tissue expression pattern of IL-34.The results showed that grass carp IL-34 was differentially expressed in all tissues,with the highest expression level in the spleen,followed by the intestine,head kidney,thymus,gill,skin and brain,and the lowest expression in the liver.Subsequently,the grass carp was infected with Flavos carp reovirus(GCRV)by intraperitoneal injection.The result of qPCR detection showed that the expression of IL-34 in the head kidney increased after 12 h after infection with Flavobacterium columnum.After 72 h,the expression of IL-34 in the spleen increased.The expression of IL-34 in the spleen and the gill after 7 d of infection with GCRV virus increased significantly,indicating that IL-34 may play an important role in the immune response of grass carp against pathogenic microorganism invasion.3.The nucleotide fragment of the grass carp IL-34 CDS region from which the signal peptide was removed was cloned into the prokaryotic expression vector pEHISTEV,the six-histidine grass carp IL-34 mature peptide fusion protein was successfully induced expressed by IPTG,and subjected to Superdex 75 column chromatography,purified IL-34 protein was obtained.The biological activity of IL-34 protein was verified by in vitro isolation of primary macrophages from grass head kidney,and the function of prokaryotic expression of IL-34 was analyzed comparatively by using the recombinant protein of grass carp IL-34 expressed in HEK-293 T cells..The results showed that IL-34 can increase the expression of proinflammatory cytokines IL-1?,IL-6 and IL-8 in macrophages,and decrease the expression of inhibitory cytokines IL-10 and TGF-?1,indicating that IL-34 can enhance the inflammatory response,may be it is a pro-inflammatory cytokine.4.The grass carp IL-34 polyclonal antibody was prepared and the specificity of the antibody was verified by Western blotting.Histochemical analysis was carried out on grass carp infected with Flavobacterium columnum for 3 d.The expression of IL-34 in the kidney and intestinal tissues after bacterial infection was quantitatively and qualitatively verified.It was found that the positive region of IL-34 in the infected state was mainly concentrated in the renal tubular cavity,while the positive region in the intestine was mainly concentrated in the the outer surface of the intestine.The above studies have learned the molecular information and expression pattern of grass carp IL-34,and have characterized the biological function of IL-34 in the grass carp immune system,enriching the study of IL-34 in teleost fish,provideing an important theoretical basis and potential application prospect for clarifying the immune regulation function of grass carp IL-34 in disease prevention and control.