The Establishment Of ELISA Method About Porcine Reproductive And Respiratory Syndrome Virus Recombinant Protein N And Preparation For Monoclonal Ntiboody

Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiration Syndrome, PRRS) is a disorder caused by breeding farms, one of the major diseases, PRRSV genome a total of six kinds of structural protein genes, respectively, from the ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7 genes encoding virus GP2, GP3, GP4, GP5, M protein and N protein, in which N protein-based advantages of the virus structural proteins, and the protein has a good response to the immunogenicity and allergenicity, for use in the diagnosis of PRRS protein of choice. In this study, a prokaryotic expression system of the PRRSV N protein gene for efficient expression of soluble, and the reorganization of the N protein as antigen, successfully established a detection of PRRSV antibodies in serum ELISA indirect method of N protein and anti-histidine tag monoclonal cell line.Clones using the genetic engineering method N protein gene Asia to the nucleus the reorganization which in expression vector PET32a constructs to express material particle PET32a-N, carries on increases the raise, succeeded under the IPTG induction expresses, has obtained the size approximately is 33KD fusion protein N-His, with anticipated result match case; The expression quantity approximately composes the bacterium total protein quantity 29%. Reorganizes the N-His fusion protein using the nickel agarose gelatin resin chromatographic analysis column purification, the purification fusion protein after the SDS-PAGE electrophoresis and Western the blot appraisal, the goal protein purity reaches as high as 91%, and has the good immunity original activeness.At the same time, using the recombinant N-His fusion protein, as coated antigen, by optimizing the reaction conditions of each step, the establishment of a pig serum can 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 163 serum were parallel clinical testing, the two masculine gender masculine coincidence rate is 83.9%, the negative coincidence rate is 84.2%, the total coincidence rate achieves 84%, indicated establishes N-ELISA has the high specificity and the sensitivity.Expression of the fusion product of N-His according to 120μg / dose only Freund's adjuvant emulsified with the contour by subcutaneous immunization BALB / c mice 3 times, from spleen cells with SP2 / 0 myeloma cells for fusion to the fusion protein N-His for the coating antigen, through the establishment of an indirect ELISA method of integration of the supernatant of cells to conduct a preliminary inspection, then the entire PRRSV virus coated ELISA method established by the second detection, screening positive clones, with three times after subcloning the stability of the secretion of an anti-histidine tag protein antibody-positive cell clone strains (3B6,4C10). Indirect ELISA detection of hybridoma supernatant titer was 1:100~1:400, while the ascites titer was1:400~1:1600; with pseudorabies virus (PRV), classical swine fever virus( HCV), porcine parvovirus (PPV) no cross-reaction; McAb hybridoma cell line for this for many times, the secretion of McAb titer of basically the same.