Abstract: The preparation and preliminary application of monoclonal antibodies (mAbs) specific to chicken Toll-like receptor 15 (ChTLR15) is described. The gene fragment encoded 162-386 amino acids of ChTLR15 were amplified by PCR and cloned into the vector pET-30a. IPTG was used to induce expression of the recombinant plasmid, the collected target protein was first purified by Ni-NTA affinity chromatography medium method, and the purified protein was renatured by dialysis using urea with concentration gradient. The renatured protein was purified again by gel filtration chromatography to obtain high-purity recombinant ChTLR15 (162-386 aa) protein. The 6-week-old female BALB/c mice were immunized with multi-loci subcutaneous injections, and the spleen cells of mice with higher serum antibody levels after immunization were fused with SP2/0 myeloma cells, and the limiting dilution method was used for multiple rounds of screening to obtain mAbs against ChTLR15 protein. The ChTLR15 gene was truncated and expressed by the prokaryotic expression system, and the ChTLR15 epitopes targeted by the mAbs were identified. The mAb 6C7 was used to locate the ChTLR15 in chicken HD11 cells with laser confocal microscopy. At the same time, the distribution of ChTLR15 in some chicken tissues was determined by immunohistochemistry. The recombinant plasmid pET-30a-ChTLR15 (162-386 aa) was successfully constructed, and the recombinant protein ChTLR15 (162-386 aa) at about 26 ku in the form of inclusion body was obtained after induced expression by IPTG. The optimal antigen coating concentration (0.35 μg·mL^-1) and the optimal serum dilution (1:6 400) were determined by indirect enzyme-linked immunosorbent assay (ELISA). A total of 4 mAb hybridoma cell lines that can stably secrete antibody against ChTLR15 were obtained, and they were named 2G4, 6C7, 6D10 and 7C4. The subclass of 2G4 and 6C7 was IgG2a, and the subclass of 6D10 and 7C4 was IgG1. Western blot results showed that the 4 mAbs can react specifically with ChTLR15, but not with other tested proteins. The prokaryotic expression system was used to express the truncated ChTLR15 (162-386 aa), and the epitopes of ChTLR15 mAbs were identified. The results showed that the epitope recognized by mAbs 2G4 and 7C4 was ²³⁰QLGTVLEF²³⁷, and the epitope recognized by 6C7 and 6D10 was ²⁴⁵EMDLLS²⁵⁰. Observation under a laser confocal microscopy suggested that ChTLR15 protein was located on the cell surface. Immunohistochemistry showed that the liver, spleen, lung, and kidney from the health birds had weak positive staining, and the positive staining of liver, spleen, lung, and kidney from the avian pathogenic Escherichia coli(APEC) challenged birds was enhanced. In this study, four mAbs against ChTLR15 protein were successfully developed and the epitopes recognized by them were identified, which is conducive to further research on the structural and functional characteristics of ChTLR15. It provides a robust tool for subsequent research in related fields such as signal pathways, immune mechanisms, and vaccine development.

Key words: ChTLR15, monoclonal antibody, antigenic epitope, immunohistochemistry, subcellular localization