The Biological Activity Of Recombinant Chicken Interleukin-9,Methods Development For Its Detection And Identification Of Its Receptor

As a member of the common y-chain receptor cytokine family,interleukin-9(IL-9)specifically binds to the IL-9 receptor a chain(IL-9Rα)on the surface of lymphocytes,mast cells,etc,producing a variety of immunomodulatory effects such as promoting cell growth and cytokine production,and then playing a variety of functions in allergic diseases,autoimmune diseases,infectious diseases and tumor immunity.Although IL-9 has a powerful immunomodulatory effect in mammals,it is not clear whether avian IL-9 has a similar function,and only chicken IL-9(chIL-9)is currently annotated in the genome.In this study,we cloned the chIL-9 gene sequence,analyzed its molecular evolution and tissue distribution,expressed the recombinant chIL-9(rchIL-9)protein and studied its biological activity in vitro and in vivo,produced polyclonal antibody(pAb)and monoclonal antibodies(mAb)against chIL-9,and established several detection methods to chIL-9.In order to further study the role of chIL-9 in diseases,mAbs against chIL-9Ra were also prepared,and the natural chIL-9Rα was identified.These results laid a foundation for further study the role of chIL-9 in immune-related diseases and the development of novel immune adjuvants in chickens.1.Recombinant expression of chIL-9 and its biological activityBased on the chIL-9 gene sequence(Genbank accession num:AM773755.1)in NCBI,Specific primers were designed,and the coding region sequence(CDS)of the chIL-9 gene was amplified using a template of peripheral blood mononuclear cell(PBMC)cDNA.The cloned chIL-9 gene consists of 417 nucleotides that encode a protein of 138 amino acids,with signal peptides in the top 20 amino acids and a theoretical size of 13.6 kDa(without signal peptides).The amino acid sequence alignment and evolutionary analysis indicated that chIL9 had less than 30%homology with mammalian IL-9 amino acids,and formed an evolutionary cluster,along with budgies,which is far away from the mammals.RT-qPCR analysis showed that chIL-9 mRNA was only abundantly distributed in the testes and thymus of chickens,and highly expressed in activated splenocytes.The signal peptide-deleted chIL-9 sequence was cloned into the prokaryotic expression vector pET-32a,and expressed in E.coli by IPTG induction as a form of inclusion body(32 kDa),and the soluble protein was obtained by purification and refolding.The full-length chIL-9 gene was cloned into the eukaryotic expression vector pEGFP-C,and transfected to DF-1 cells to obtain a secretory rchIL-9(1525 kDa),and it was found that eukaryotic rchIL-9 was an N-glycosylated protein when treated with deglycosylase.The biological activity of rchIL-9 was measured by co-culture with monocytes/macrophages or PBMCs,and it was found that rchIL-9 activated monocytes/macrophages,promoted the proliferation of CD3+T cells.Furthermore,rchIL-9 also improved the level of nuclear transcription factor PU.1 and inflammatory factor IL-1βmRNA in PBMC,splenocytes and thymocytes,and also regulated the expression of IFN-γ,IL-2,IL-7 and IL-9 mRNAs.Those results illustrated that prokaryotic or eukaryotic rchIL-9 is biological activity,and also has the potential to promote the differentiation of chicken Th9 cells.2.Biological activity and effects of recombinant chIL-9 in vivoIn order to measure the biological activity of chIL-9 in vivo,different doses of prokaryotic rchIL-9 were used to treat chicks,and the body weight gain of chicks was analyzed.Multicolor flow cytometry and RT-qPCR were used to analyze the dynamic T cell subsets changes of peripheral blood and spleen mononuclear cells and their cytokine mRNA changes at different time points.The results showed that rchIL-9 did not affect the weight gain rate of chickens,but induced an increase in the number and percentage of CD4 T cells and a decrease in the number and percentage of CD8 T cells in peripheral blood.After rchIL-9 treatment,the number and percentage of γδ T cell subsets(CD8α+,CD4-CD8-,CD4+)was increased to varying degrees at different time points,and the number of natural killer(NK)cells in peripheral blood and spleen was decreased in the early stage of treatment and increased in the later stage.The qPCR results showed that rchIL-9 downregulated PBMCs and splenocytes IL1β mRNA,and upregulated PBMCs and splenocytes PU.1 factor mRNA level,and also regulated IFN-γ,IL-2 and IL-7 mRNAs.These results suggested that rchIL-9 has the function in promoting the expansion of CD4 T cells and regulating cytokine expression in vivo.3.Generation of pAb and mAbs against chIL-9 and establishment of its detection methodsThe prokaryotic rchIL-9 was used as an immunogen to generate rabbit anti-chIL-9 pAb(titer,1:2×105)and 14 mouse mAbs against chIL-9,named 1C7,1D4,1D7,1F7,1F10,2B2,2B12,3H5,4F7,5B3,5B8,5B9 and 5D2,respectively.Among these mAbs,mAb 1D4 and 1F7 belong to IgG2a,the other mAbs belong to IgG1 subtype.All the mAbs have κ-light chain of immunoglobulin.The titer of ascites prepared was between 1:105-107.Immunocytochemistry(ICC)and Western blot(WB)results showed that all 14 mAbs specifically recognized eukaryotic or prokaryotic rchIL-9.Further identification found that rabbit pAb and mAb 3H5,4F7,4H7 and 5B8 well recognized natural chIL-9 as well as mature chIL-9 secreted outside the cell,with a size of 25 kDa.Flow cytometry analysis found that all 14 mAbs could detect eukaryotically expressed rchIL-9 protein by intracellular cytokine staining,but only mAb 1C7,1F10,and 5D2 could specifically detect native chIL-9 expressed in activated splenocytes with a frequency of only 0.14%.The chIL-9 antigen capture ELISA was developed by using 5B8 mAb as the capture antibody and biotinylated 1F7 mAb as the detection antibody.The optimal conditions for the test were 10 μg/mL for 5B8 mAb;The 5B8 mAb was coated at 4℃ for 12 h;0.5%BSA was used as blocking solution and detection antibody diluent;The biotinylated 1F7 mAb was used at 1 μg/mL.The standard curve was established with the detection limit of 1.5 ng/mL and an approximate linear relationship in the interval of 3-100 ng/mL.The established method does not cross-react with chicken IL-4,TGF-β1,IFN-γ,or IL-2.This method was used to detect the content of rchIL-9 in the supernatant of eukaryotic transfected DF-1 cells,which reached 400 ng/mL,and chIL-9 reached 12 ng/mL in the PMA/Ionomycin-activated splenocytes(4×107 cells,48 h)supernatant.The generation of pAb and mAbs against chIL-9 and the establishment of chIL-9 detection methods provide a tool basis for further study of the role of chIL-9 in diseases.4.Generation,identification of mAbs to chIL-9 receptor α chainTo assist in studying the role of chIL-9 in diseases,we also cloned and analyzed chIL9Rα,and its mAbs were also prepared.According to the predicted chIL-9Rα sequence(GenBank accession num:416587)provided by NCBI,primers were designed,and the fulllength sequence and the extracellular region sequence were amplified,respectively.Amino acid sequence analysis found that chIL-9Rα had less than 25%homology with mammalian IL-9Rα,forming an evolutionary cluster far from mammals alone.RT-qPCR results showed that chIL-9Rα mRNA was only abundantly expressed in heart and activated PBMCs.The extracellular region sequences of chIL-9Rα were cloned into prokaryotic expression vector pET-28a and eukaryotic expression vector pcDNA3.1 respectively to obtain prokaryotic and eukaryotic recombinant proteins.The prokaryotic chIL-9Rα was used as an immunogen to generate 11 mAbs against chIL-9Rα,named 1D9,2D9,2G5,3H6,4E1,4E4,4E6,4F5,4G10,4H10 and 5G5,respectively.Among these mAbs,mAb 1D9 belongs to IgG2a,the other mAbs belong to IgG1 subtype.All the mAbs have κ-light chain of immunoglobulin.ICC and WB results showed that all mAbs except for mAb 4H10 specifically recognized the recombinant chIL-9Rα.Indirect immunofluorescence assay(IFA)found that only 2G5 and 5G5 mAbs recognized chIL-9Rα on the surface of splenocytes.WB results showed that chIL-9Rα was also present on the surface of the MSB-1 cell line,with a molecular weight of 51 kDa.The recombinant chIL-9Rα protein and anti-chIL-9Rα mAbs provides immunological tools for further study of the function and mechanism of chIL-9.