Preparation Of Monoclonal Antibody Against PRRSV-NP And Its Establishment Of Direct Immunofluorescence Assay Technique

Porcine reproductive and respiratory syndrome(PRRS),which is caused by porcine reproductive and respiratory syndrome virus(PRRSV),leads to serious infectious diseases and reproductive disorders such as abortion,weak piglets,stillbirth,mummy fetus,and causes huge economical losses to swine industry.PRRSV grows mainly in porcine alveolar macrophages,monocytes and other immune cells,and damage the immune function of pigs.While the virus can exist for nearly 16 months in vivo,resulting in immunosuppression and co-infectons with other viruses.Especially,co-infection of PRRSV and PCV2 is very common in pig farms,which seriously affects the reproductive performance of sows and the survival rate of piglets.Therefore,effective and accurate diagnosis of PRRSV is of great significance to detect PRRSV latent infection and co-infection and PRRS control of pig industry.PGEX-6P-PRRSV-N plasmid was constructed to express PRRSV-N protein,and mice were immunized with purified PRRSV-N protein.Hybridoma cell was obtained by the way of cell fusion,and two positive hybridoma cells N-13 and N-29 was obtained according to the established indirect ELISA assay.Mice were immunized with positive hybridoma cells and mice ascitic fluid was prepared,while the ELISA titer of N-13 is 1:10000 and N-29 is 1:12000.N-29 antibody with high titer was selected and identified by indirect immunofluorescence assay and indirect ELISA method.The results showed that there was no cross reaction between N-29 antibody and porcine pseudorabies virus(PRV),porcine diarrhea virus(PEDV)and porcine circovirus(PCV).Western blot result indicated that monoclonal antibody has good immunogenicity.Therefore,monoclonal antibody can be used for clinical detection of PRRSV,and used for immunofluorescence assay,competitive ELISA and immunochromatography assay.In order to establish a rapid method for diagnosis of PRRSV,BALB/C mice were immunized with purified recombinant PRRSV-TJ-PGEX-6P-N protein as antigen.PRRSV-N protein with His tag was coated by using lymphoma cell fusion technology,two strains of monoclonal antibodies against N protein was screened by indirect ELISA method,named as N-13 and N-29.They are identified by indirect ELISA method,western blot and direct immunofluorescence assay(DFA)and its affinity coefficient was determined.The titer of ascites were 1:10000 and 1:12000,ascites N-29 with high titer was purified and labeled with antibody markers.The sensitivity,specificity and preservation period of labeled antibody were identified.Then,a sensitive DFA diagnosis method was established through the optimization of DFA conditions.Compared with the traditional PRRSV identification method by CPE observation,DFA is a more accurate and can be used as an effective method for PRRSV isolation and identification in the laboratory and PRRS diagnosis in swine industry.