Production And Identification Of Monoclonal Antibody Against Recombination N Protein Of PRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) is contagious diseases virus as the main features of pregnant sows with premature birth, miscarriage, stillbirth, mummy embryo and weak piglets with piglet coughing, difficulty breathing and shortness of breath. Now, PRRSV is mainly American-type virus in China. At present, the main detections of this disease are serological test, ELISA method and real-time fluorescence quantitative PCR assay. These methods dependent on laboratory diagnosis what is complex, time-consuming, polluting, and needs expensive equipment. Colloidal gold immunochromatographic technology does not need a professional and technical staff.It is a fast, simple, low-cost detection method with a small amount of sample. This study is about gene cloning, prokaryotic expression and purification of recombinant protein of the conservative nucleocapsid protein N of PRRSV. Then the N protein was obtained with high purity as a specific antigen, to filter out specific, high titer and stability monoclonal antibodies of the N protein from secreting anti-PRRSV monoclonal antibody-positive hybridoma cell, and purity of the monoclonal antibody ascites. This study is to lay the material foundation for the next step.of Colloidal gold immunochromatographic test strip.This study designs a pair of specific primers according to GenBank published ORF7of PRRSV VR2332strain of full-length gene sequence.And it applied RT-PCR amplification of a complete ORF7gene, the gene fragment to be372bp (encode nucleocapsid protein of PRRSV). The PCR product was cloned into pMD-18T vector for sequencing. After sequencing the fragment is connected to the prokaryotic expression vector pET-28a(+) to build the prokaryotic expression plasmid pET-28a(+)-N. To optimize the conditions on the induced expression (IPTG concentration, induction time, induction temperature) the results can be realized recombinant N protein highly expressed in37℃,0.2mmol/L IPTG induction5h.The expression of recombinant N protein molecular weight is about22ku, as same as the molecular weight of theoretical speculation of the protein. After the recombinant N protein purified by Ni-NTA affinity chromatography, it is showed that recombinant N protein specificly react with PRRSV positive serum by Western-blotting analysis. The purified recombinant N protein is used as immunogen, according to long-term immunization to immunize BALB/c mice. The spleen cells from immunized mice interfuse with SP2/0myeloma cells. With purified PRRSV antigen for the ELISA, it filters a monoclonal antibody hybridoma named by the McAb-A7B1. The results of ELISA and Western-blotting analysis show that supernatant titer of this cell line is1:128, ascites titer is1:10s. It has no cross reaction with PRV, PPV and JEV. It is showed that the monoclonal antibody of McAb-A7B1is high titer and good specificity.