Preparation and identification of a monoclonal antibody against N protein of porcine deltacoronavirus

Abstract:

[Background] Porcine deltacoronavirus (PDCoV) is a newly discovered porcine enteric coronavirus, which causes gastrointestinal disease characterized with acute watery diarrhea, vomiting and dehydration, etc. Among the four structural proteins of PDCoV, nucleocapsid protein (N) is a highly conserved structural protein, which plays an important role in pathogen diagnosis. [Objective] A monoclonal antibody (MAb), based on purified recombinant PDCoV N protein was prepared and its characteristics were systemically identified. [Methods] The constructed recombinant pET-28a-N bacteria was induced by IPTG for N protein expression, and BALB/c mice were immunized with purified N protein to prepare hybridoma cells. After three rounds of subcloning screening, one hybridoma cell with the highest antibody titer was selected for the subsequent production of mice ascitic fluid. The mice asctic fluid was purified by Protein G affinity chromatography column. The monoclonal antibody were systematically identified by titer determination, subtype identification and Western blot. The diagnostic application potential of the monoclonal were evaluated by using cell indirect immunofluorescence assay, tissue paraffin section fluorescence experiments, immunohistochemistry, and flow cytometry. [Results] A hybridoma cell was screened and named as 4E88, the titer of the ascites the MAb was 1:105, the subtype was IgG1 and the light chain was κ chain. Western blot analysis showed that the MAb could specifically reacted with recombinant N protein and ultracentrifuge purified PDCoV whole virus. In addition, the MAb 4E88 can be used for indirect immunofluorescence assay, immunohistochemistry and flow cytometry for PDCoV detection. [Conclusion] The MAb 4E88 has good reactivity in this study which laid a good foundation for applied research associated with PDCoV identification, diagnosis and N protein function.

Key words:Porcine deltacoronavirus, N protein, monoclonal antibody, Preparation, Identification, Application