Abstract Heat shock protein (DnaK) is a highly conserved stress protein produced by organisms under the influence of unfavorable factors, it has dual roles of carrier and adjuvant, however, the DnaK protein of Mycoplasma synoviae (MS) is less studied. In this study, in order to prepare a monoclonal antibody (MAb) of MS DnaK protein and preliminarily characterize its antigenic epitope, the recombinant plasmid was constructed and the recombinant protein was expressed. The recombinant protein of Mycoplasma synoviae, DnaK (rMSDnaK protein) was purified. BALB/c mice (Mus musculus) were immunized with rMSDnaK protein. Mice with high antibody levels were selected, their spleens were isolated, and the collected splenocytes were fused with myeloma cells (SP2/0 cells), after which positive hybridoma cells in good condition were screened by limited dilution and indirect ELISA, and ascites were collected and purified after injection into the peritoneal cavity of quinacrine mice. The rMSDnaK was successfully expressed identified by SDS-PAGE. Indirect ELISA was used to screen one hybridoma cell line, named 6G6, which could secrete anti-DnaK antibody and had good stability. The MAb 6G6 subtype was identified as IgG3 subclass. The potency of the purified MAb was determined by indirect ELISA to be 1:102 400. Western blot results showed that MAb 6G6 reacted only with rMSDnaK protein. Immunofluorescence assay (IFA) results showed that MAb 6G6 reacted fluorescently only with MS. Western blot results demonstrated that the prepared truncated MS DnaKΔ3-3 was able to react with rMSDnaK protein, which proved that the antigenic epitope region recognized by the monoclonal antibody was amino acids 506~596 at the C-terminus. In this study, the anti-MS DnaK MAb was successfully prepared and its antigenic epitope was identified, which provides biological materials for the subsequent establishment of MS detection by double-antibody sandwich ELISA for MS detection.
LIU Kai,ZHAO Yun-Hai,HE Xiao-Xiao, et al. Preparation of Monoclonal Antibody Against DnaK Protein of Mycoplasma synoviae and Preliminary Identification of Its Epitopes[J]. 农业生物技术学报, 2025, 33(3): 689-696.
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