The Establishment And Application Of Fast Immune-detection Method Based On Nucleocapsid Protein Of HPPRRSV

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in Europe and inthe US in the early1990s and has since become a problem to the swine industry world wide.PRRSV causes a persistent and sometimes severe disease that is characterized by respiratoryproblems, weight loss and poor growth performance.In this study, recombinant strain BL21(DE3)(pET-N) was resuscitated by LB medium.The plasmids（pET-N） were isolated and verified by double-enzyme cleavage method. Therecombinant strain BL21(DE3)(pET-N) was induced in6hours under the optimizedconditions to acquire the high expression of N protein. The N protein, which used the nickelagarose resin chromatographic analysis column for the purification, was made an appraisal ofWestern blot. The results showed: the goal protein purity reached as high as90%, and Nprotein had the good immunogenicity and specificity.By the optimization of the concentration of serum,the concentration of antigen, thecoating condition, the inoculated solutions and concentration, and the concentration of Rabbitanti pig IgG, an indirect ELISA was established for detecting antibodies against N protein inswine serum. The result showed: the critical value of ELISA was0.2198with good sensitivityand specificity, and quality guarantee period of the Kit was about3month.The N protein was emulsified with Freund’s adjuvant by subcutaneous immunizationBALB/c mice. When the serum titer was50000, a cell fusion experiment was conducted byindirect ELISA. At last, we got2anti-histidine tag protein antibody-positive cell clone strainsand9anti-N protein antibody-positive cell clone strains.All of9antibodies purified was IgGand could produce immunological reactions with PRRSV in commercial ELISA Kit.9antibodies had no cross reaction with some viruses.Colloidal gold with about20nm particle diameter was prepared for the optimization ofpH of McAb, the content of McAb, and the concentration of Rabbit Anti N protein PAbs andRabbit Anti rat PAbs. Rabbit Anti N protein were micro-sprayed onto a nitrocellulosemembrane at a position that would become the capture test band (T).and Rabbit Anti rat PAbs was micro-sprayed onto the same nitrocellulose membrane at a position that would becomethe control band (C). The strip assembled according to literature in recent years could pavethe way for rapid detection of PRRSV.