Development Of Monoclonal Antibodies Specific To Chicken Toll Like Receptor 15 And Identification Of The TLR15 Epitopes Recognized By The Monoclonal Antibodies

Toll-like receptors is a kind of highly conservative type I transmembrane glycoprotein pattern recognition receptors,which can recognize a variety of pathogen associated molecular patterns,quickly initiate effective immune responses,and plays an important role in the anti-infection process of the host.In 2006,Higgs et al.conducted bioinformatics analysis on the chicken genome and found a chicken-specific gene located on the third chromosome,then designated it as Chicken Toll Like Receptor 15(ChTLR15).So far,there has been no report on ChTLR15 monoclonal antibodies against ChTLR15 except for the preparation of ChTLR15 polyclonal antibody by Bojian Xing et al.Therefore,it is of great significance to develop the monoclonal antibody against ChTLR15.In this study,primers were designed according to the ChTLR15 gene sequence deposited in GenBank,and the gene coding the extracellular region sequences(162-386 aa)were amplified by PCR.The recombinant plasmid pET-30a-ChTLR15(162-386 aa)was constructed.The recombinant plasmid was introduced into host strain BL21(DE3),and IPTG induction showed that ChTLR15(162-386 aa)-His was mainly expressed in inclusion body with a protein size of about 26 kDa,which was subsequently renatured by urea gradient dialysis.Recombinant ChTLR15(162-386 aa)-His protein was purified by Ni-NTA affinity chromatography and gel filtration chromatography,and 6-week-old female Balb/c was immunized with the recombinant ChTLR15 by subcutaneous multi-site injection.The optimal antigen coating concentration(0.35 μg/mL)and the optimal serum dilution(1:6400)were deterimined by indirect enzyme-linked immunosorbent assay(ELISA).The mice were immunized for three times and their antibody titer was determined by the established indirect ELISA.After the antibody titer reached more than 1:100000 determined in ELISA,the spleen cells of immunized mice were collected and fused with myeloma cells SP2/0.Four hybridoma cell lines,named 2G4,6C7,6D10 and 7C4,secreting monoclonal antibodies(MAbs)against chTLT15 stably,were obtained after three times of subcloning.The specificity of the four MAbs was identified by Western blot,and the results showed that all the four MAbs could react with immunogen protein.Both indirect ELISA and indirect immunofluorescence(IFA)were employed to screen the MAbs at the same time.The ELISA results showed that MAbs 6C7 and 6D10 had strong reactivity,and MAbs 2G4 and 7C4 had weak reactivity.The results of IFA test showed that MAbs 2G4,6C7 and 7C4 had fluorescence reaction with the chicken macrophage line HD11 and chicken fibroblast cell line DF-1,but did not have the fluorescence reactivity with the human emborynic kidney cell(HEK293T)transiently expressed chTLT15.Mab 6D10 recognized HEK293T transiently expressed chTLT15,but did not recognize HD11 and DF-1 in IFA.The results showed that the ELISA titers of MAbs 6C7 and 6D10 ascites were 1:20480 and 1:40960,respectively,but there were no ELISA titers for MAbs 2G4 and 7C4 ascites.The IFA titers of MAbs 6C7 and 7C4 in HD11 and DF-1 cells were 1:2560 and 1:5120,respectively,while Mab 2G4 was lower,and Mab 6D10 had no IFA titer in the above cells;The IFA titer of MAb 6D10 in HEK293T cells transiently expressed chTLT15 was 1:320.Subclass identification was performed on the supernatants of the four MAbs according to the monoclonal antibody subclass typing kit,and the results showed that subclass of MAbs 2G4 and 6C7 were IgG2a,and subclass of MAbs 6D10 and 7C4 were IgG1.To determine the epitope recognized by the MAbs,a series of truncated ChTLR15 were employed to react with the MAbs in Western blot.The epitope recognized by MAbs 2G4 and 7C4 was 230QLGTVLEF237,and the epitope recognized by MAbs 6C7 and 6D10 was 245EMDLLS250.Accurate epitope identification is helpful for understanding of the immune response and the robust application of MAbs.Immunohistochemical analysis using MAb 6C7 showed that the expression of TLR15 in liver,spleen,lung and kidney in infected chickens was significantly upregulated compared with those in the mock chickens.The localization of ChTLR15 in HD11 cells was achived by laser confocal technique using MAb 6C7,and the results showed that ChTLR15 was embedded in the cell membrane of HD11 cells.The MAbs developed in this study have good specificity and reactivity,which provides a robust tool for the study of the immune regulation mechanism related to ChTLR15,and also lays a foundation to develop anti-infection biological agents.