Identification And Functional Analysis Of Interleukin-15(IL-15) And IL-2R? In Grass Carp

Cytokines are small-molecule proteins that are secreted outside the cell and regulate immune functions by binding to corresponding receptors,and play a key role in the body's innate and adaptive immunity processes.Interleukin(IL)-15 belongs to the common cytokine receptor ? chain family,and is a pleiotropic cytokine that acts on the innate and adaptive immune system.It is very important in regulating various life activities of NK and T cells,including their appreciation and differentiation.Although IL-15 and IL-2 have low sequence homology,they are structurally related and have overlapping but different functions.Although many different cell types can express IL-15(m RNA),the IL-15 protein is mainly activated by dendritic cells(dc)and monocytes in response to Toll-like receptors(TLRs)and interacts with Body combination.The IL-15 receptor consists of three subunits ?,? and ?.IL-15R? presents IL-15 to target cells expressing IL-15R? and ?c subunits for signal transduction,and then exerts its immune function.Mammalian studies on the function and biological activity of IL-15 have been thorough,but there is very little data on the function of IL-15 and its receptors in teleost fish.In this study,grass carp(Ctenopharyngodon idella)was used as the research object to further investigate IL.-15 and its receptor expression and function.In this study,the SMART-RACE method was used to clone and identify the IL-15 and IL-2R? genes.The full-length c DNA of grass carp IL-15 was 1040 bp and the CDS was 576 bp.It encodes 191 amino acids(aa),including 161 bp 5 ‘untranslated region(UTR)and a 303 bp 3 'untranslated region.IL-15 protein has a signal peptide(1-16 a a),but no transmembrane domain,suggesting that it can be secreted by the traditional ER-Golgi pathway.The IL-2R? c DNA is 2215 bp in length,containing 1611 bp of CDS encoding the protein,5?-UTR of 87 bp,and 3?-UTR of 517 bp.1-26 aa is the predicted signal peptide,302-330 aa is the predicted transmembrane region,and the rest is the cytoplasmic region.IL-15 and IL-2R? have high homology with zebrafish sequences,which are 52.9% and 65%,respectively.Phylogenetic tree and gene isolinear analysis indicate that IL-15 and IL-2R? are highly conserved in evolution.Analysis of IL-15 and IL-2R? expression in various tissues of healthy grass carp by q PCR.The results showed that IL-15 and IL-2R? were constitutively expressed in healthy tissues of grass carp,and both were highest in the spleen,followed by gills,brain,intestine,and skin,with the lowest expression in the head kidney and liver,respectively.Infection of grass carp with Flavobacterium columnare(F.bacillus)and reovirus(GCRV)through intraperitoneal injection,q PCR results showed that the expression levels of IL-15 and IL-2R? were up-regulated in the spleen and kidney tissues of 1 day,2 days,and 3 days after infection with grass carp by FC,After reovirus infection of grass carp,the expression levels of IL-15 and IL-2R? in the spleen and kidney tissues were also significantly up-regulated at 7 and 14 days after infection.The above results indicate that grass carp IL-15 and IL-2R? participate in the host's immune defense response against bacterial and viral infections.In order to further clarify the immunological function of IL-15,the mature peptide IL-15 and IL-2R? fragments were inserted into the prokaryotic expression vector PET-21 d,and the fusion protein of grass carp IL-15 and IL-2R? was successfully expressed by IPTG induction.Purified IL-15 and IL-2R? were obtained by gel column chromatography(Super75dex).Grass carp primary head kidney leukocytes were isolated in vitro and incubated with prokaryotic expressed IL-15 protein to verify the activity of IL-15 protein.The results showed that IL-15 could significantly increase the expression of IFN-?,and had no significant effect on the expression of other inflammatory factors.IFN-? is produced by activated T cells and NK cells,indicating that IL-15 can promote the activation of T cells and NK cells.At the same time,prokaryotic expressions of grass carp IL-4 / 13,IL-2,IL-21 and IFN-? were incubated with head and kidney cells to verify the regulation of IL-15 by cytokines.The results show that IFN-? can increase IL-15 expression.The above studies identified grass carp IL-15 and IL-2R? and conducted bioinformatics analysis.After F.bacillus and reovirus infection,their expression profiles were analyzed,and the role of IL-15 in the grass carp immune system The function was explored,which enriched the research on IL-15 in osteochondria,provided a theoretical basis for the in-depth description of the immune regulation function of IL-15,and provided a potential application prospect for fish disease prevention and control.