Summary auto-generated
This study describes the generation and epitope mapping of 36 monoclonal antibodies (MAbs) specific for rabies virus phosphoprotein P. The P protein was expressed as a recombinant protein in E. coli from the PV strain and used to immunize mice. Resulting MAbs were characterized by ELISA, Western blotting, and immunofluorescence assays, where they detected cytoplasmic inclusions in infected cells. Seven MAbs were further analyzed for epitope mapping using truncated P protein fragments expressed in transfected cells. The mapping revealed five epitope groups distributed across different protein domains. Notably, 60% of MAbs recognized a major antigenic determinant located between amino acids 83–172, suggesting this region is immunodominant. Most epitopes consisted of linear sequences rather than conformational structures. These MAbs represent the first comprehensive panel against rabies virus P protein and provide tools for investigating the structural and functional properties of this multifunctional regulatory protein involved in viral transcription and replication.
Key findings
- Thirty-six monoclonal antibodies were successfully generated against rabies virus P protein using recombinant protein expressed in E. coli
- Epitope mapping revealed five distinct epitope groups; 60% of MAbs recognized a major antigenic site in the central region (amino acids 83–172) of the P protein
- Most antibody epitopes consisted of linear amino acid sequences rather than conformational structures, as demonstrated by reactivity in denaturing conditions
- Specific MAbs were mapped to carboxy-terminal regions (222–297), amino-terminal region (1–19), and internal domains, with notably no antibodies recognizing residues 138–222
- These MAbs provide valuable tools for studying protein-protein interactions within viral complexes and distinguishing between rabies virus strains based on amino acid differences
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Abstract
Thirty-six monoclonal antibodies (MAbs) specific for the rabies virus P phosphoprotein were obtained from mice immunized with recombinant P (PV strain) produced in E. coli. All MAbs reacted against the corresponding rabies virus protein by ELISA and by Western blot analysis and revealed the presence of cytoplasmic inclusions in rabies virus infected cells. The epitopes of seven MAbs were mapped by testing their reactivity with protein fragments expressed from deletion mutants in transfected cells. Western blotting, immunoprecipitation and immunofluorescence assays were performed. These MAbs recognized epitopes in different domains of the P protein: 60% were directed against a region lying between residues 83-172 suggesting a major antigenic determinant of the rabies virus P protein in this region. Most of the antigenic sites appeared to be composed of linear epitopes. These MAbs will be useful as tools to dissect structural and functional properties of the rabies virus P protein.