Identification of the B-cell epitopes on N protein of type 2 porcine reproductive and respiratory syndrome virus, using monoclonal antibodies

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (N) is the immunodominant region of PRRSV viral proteins. However, B cell epitopes present on N protein have not been well characterized. In this study, The ORF7 gene was amplified by RT-PCR and inserted into the expression vector pET-28a, the constructed pET-28a-N was transformed into Escherichia coli BL21. After expression, purification and identification, the recombined N protein was used as the target to generate monoclonal antibody (mAb). Strains of hybridoma cells secreting anti-N mAbs were obtained by the hybridoma technique. Three of them (named 1G4, 1C6, 3D11) were specifically reacted with PRRSV by IPMA and IFA. To identify the B-cell epitopes within PRRSV N protein, six serial overlapping synthesized peptides (P1-P6) covering the whole region of N were used to define the epitopes recognized by 1G4, 1C6, 3D11. Importantly, after the identification of dot-ELISA and indirect ELISA, we found that 1-15aa was a new epitope which had never been reported before and that it was highly conserved among some HP-PRRSV isolates of type 2 PRRSV. The results of this study might open new perspectives on the detection of PRRSV and have important implications for studing the structure of the N protein.