Expression Of The Major Core Protein N Of PRRSV In Eukaryotic,Establishment Of ELISA And Preparation Of Monoelonal Antibodies

Porcine reproductive and respiratory syndrome characterized by severe reproductive disorders in sows and respiratory diseases in young pigs,was the first recognized in the US in 1987.Since its first appearance.PRRS has been causing immense economic loss in swine industry all over the world.As the most abundant protein of PRRSV,the nucleocapsid protein is highly antigenic.That makes it be a suitable candidate for diagnosis and detection. Purpose of this experiment was as follows research.The experimental parts studied on N gene of PRRSV. N gene of PRRSV was cloned into pFastBacHTB plasmid of Bac-To-Bac expression system. Bacmid-N was obtained, through homologous recombination between pFastBacHTB-N and bacmid. After transfection, recombined virus was developed. The specific 17KD band was showed by analysis of SDS-PAGE and Western blot, which indicated that PRRSV N protein had expressed in insect SF9 cells.Using the recombinant N-His fusion protein,as coated antigen,by optimizing the reaction conditions of each step,the establishment of a pig serumcan be detected from porcine reproductive and respiratory syndrome recombinant N protein antibody ELISA indirect method.And established methods and N-ELISA imported PRRS detection kit IDEXX-ELISA of 263 serum were parallel clinical testing,the two masculine gender masculine coincidence rate is 98.4%,the negative coincidence rate is 97.2%,the total coincidence rate achieves 98.1%,indicated establishes N-ELISA has the high specificity and the sensitivity.Monoclonal antibodies (McAbs) 3D12,4E10,6E2 and 7G6 against PRRSV were produced by fusing SP2/0 myeloma cells with spleen cells of Balb/c mice immunized with purified PRRSV. The indirect ELISA results showed that the antibody titers of cell supernatant were 1:256,1:512,1:512 and 1:256. The titers of ascites were 1:1.024×106,2.048×106,1:1.024×106 and 1:2.048×106. The result of ELISA showed that all the McAbs reacted with PRRSV but not with other virus.