Abstract: In this experiment, the nucleoprotein (N) gene of porcine reproductive and respiratory syndrome virus(PRRSV) amplified by RT-PCR was cloned into pet-30a vector and expressed in Escherichia coli BL21, then induced with IPTG. The expressed protein was identified by SDS-PAGE and confirmed by Western blotting. The recombinant N protein was purified by Ni. The BALB/C mice were immunized through NC filter immunization, the splenocytes of the immunized mice were fused with SP2/0 cells, the hybridoma cells were screened and confirmed to prepare monoclonal antibodies. The SDS-PAGE and Western blotting showed that the recombinant N protein was 20 ku in size and specifically reacted with anti-PRRSV positive serum. After purifying in Ni, the recombinant N protein was shown as one specific band in SDS-PAGE. The NC filter immunization was a good method. After cells fusion and screening, three hybridoma cells which produced McAbs steadily were screened by ELISA, named H7, F7, C8. the special anti-nucleocapsid protein monoclonal antibody-H7 was prepared. IFA and Western blotting assays showed that the monoclonal antibody reacted with PRRSV N protein specially. The McAb belong to IgG2b. The chromosomes analysis of the hybridoma cells confirmed the correct number of chromosomes. Finally,the procaryotic expression and purification of recombinant N protein and its highly specific monoclonal antibody were successfully achieved. This sudy laid the foundation for the analysis of N protein.

Key words: PRRSV; nucleocapsid protein; procaryotic expression; purification; monoclonal antibody