Procaryotic Expression and Purification of the Recombinant Nucleocapsid Protein of PRRSV and Preparation of the Monoclonal Antibody

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.