Preparation Of Monoclonal Antibodies Against Chicken Toll-like Receptor 15 And Their Preliminary Application

Toll-like receptors(TLRs)are important pattern recognition receptors(PRRs)of the innate immune system,which recognize a variety of highly conserved pathogen-associated molecular patterns(PAMPs)and initiate innate and adaptive immune responses.Toll-like receptor 15(ChTLR15)appears to be unique to avian species and has a specific and unique role in defense against avian pathogens at the molecular level unlike other known TLRs.Currently,the ligand for ChTLR15 is unknown,and the known classical agonists of TLRs do not activate ChTLR15.To date,no reports on the development of monoclonal antibodies(MAbs)against the natural ChTLR15 at 127 kDa have been published.The aim of this study is to develop MAbs against natural ChTLR15 to support the in-depth study of the biological function of ChTLR15.The open reading frame of the ChTLR15gene sequence harbours 2607 nucleotides and encodes 868 amino acids,according to GenBank.TMHMM,Bepipred software were used to predict ChTLR15 transmembrane structural domains and linear epitope regions.The concentration,purity and reactivity of ChTLR15(162-386 aa)preserved in liquid nitrogen in our laboratory were identified by Bicinchoninic acid(BCA)assay,SDS-PAGE and Western blot,respectively.6-week-old BALB/c female mice were immunized twice with qualified ChTLR15(162-386 aa),and an optimal coating concentration of 3 μg/mL ChTLR15(162-386 aa)was determined in an indirect enzyme-linked immunosorbent assay(ELISA).Mice with an antibody ELISA titre of 1:25600 were selected for booster immunization before fusing their splenocytes with myeloma cells SP2/0.Positive hybridoma cell lines were screened by ELISA,Western blot and immunofluorescence assay(IFA).Three hybridoma cell lines,named 3G9,5B10 and 5C7,were obtained by three three cycles of subcloning,which could stably secrete antibodies.All three MAbs experienced strong ELISA reactivity and fluorescene reactivity with chicken-origin HD11 and DF-1 cells evaluated by IFA,MAb 3G9 with the strongest IFA activity among three MAbs,and their subclasses were IgG2b,IgGl and IgG1,respectively.The MAbs 5B10 and 5C7 could recognize naive ChTLR15 with a molecular weight of 127 kDa originated from HD11 cells,and MAbs 3G9 and 5C7 reacted with subunits of ChTLR15 with molecular weights of about 35 and 95 kDa from HD11 cells in Western blot,respectively.The ELISA titres of MAbs 3G9,5B10,and 5C7 ascites were 1:102400,1:51200,1:51200,respectively.It was found that ChTLR15 located on the cell membrane of chicken DF-1 cells stained with FITC-MAb 5B10 under a confocal laser scanning microscope(CLSM).In vitro infection of macrophages showed that ChTLR15 expression was significantly enhanced after live and inactivated avian pathogenic Escherichia coli(APEC)E516(O1 serogroup),E058(O2 serogroup)or E522(O78 serogroup)strain infection from 2 to 18 h in HD11 and chicken peripheral blood mononuclear cell(PBMC)cells,while lipopolysaccharide(LPS)was unable to activate its expression;In HD 11 cells,either APEC or LPS upregulated the transcription level of both ChTLR15 and its downstream signaling pathway-related factors MyD88,NK-κB,and IL-1β.In vivo infection experiments demonstrated that ChTLR15 transcription levels in APEC(E516,E058 strains)-infected 1-day-old specific pathogen free(SPF)chickens were very significantly upregulated in the spleen,cecum,and bursa,and significantly upregulated in the thymus,liver,pancreas,and bone marrow,and no transcription level change was observed in the heart,kidney,lung,and brain of challenged birds.Immunohistochemistry(IHC)results showed that after APEC challenge,ChTLR15 expression was enhanced in all tested tissues of challenged birds except for the brain,and positive cells in the cecum,bursa of Fabricius,and spleen presented as dark yellow staining,for those of liver,pancreas,lung,kidney,and heart were stained as light yellow.In summary,we successfully prepared MAbs against natural ChTLR15 receptor,which can be used for IHC,IFA,Western blot and ELISA,providing an important and necessary tool for further study on both biological function of ChTLR15 and interaction between pathogen and host,and confirming APEC triggers both the ChTLR15 and its downstream signal pathway activation,leading to deepen our understanding on innate immune mechanism of birds dealing with the APEC and other pathogens infection.