Preparation Of The Monoclonal Antibodies Against Nucleocapsid Protein Of Porcine Reproductive And Respiratory Syndrome Virus And Their Potential Application

The BALB/c mice were immunized intraperitoneally with the BacN, which was the expression product of the nucleocapsid protein gene of porcine reproductive and respiratory syndrome virus (PRRSV) in Sf9 cell with Bac-to-Bac baculovirus expression system. Three weeks later, the mice were immunized intraperitoneally with the same dose for the second time and after another two weeks the mice were immunized intraperitoneally with twice the dose for the final immunization. Three days later, spleen cells from immunized mice were fused with SP2/0-Ag-14 myeloma cells. Indirect enzyme linked immunosorbent assay (iELISA) was used to screen hybridoma cells, and limiting dilution method was applied to subclone positive hybridoma cells three times, and three positive clones, designated as 1F9, 5A5 and 5H8 respectively, were obtained from hybridoma cell lines. The titers of their ascitic fluids were 215, 217 and 218 in iELISA and 216, 216 and 215 in indirect immunofluorescent assay(IIF) , respectively. They were subtyped to be IgGl, IgG2b and IgG2b, respectively. It was verified that these 3 monoclonal antibodies (MAbs) reacted with PRRSV while did not react with porcine parvovirus (PPV) , pseudorabies virus (PRV) , Japanese encephalitis virus (JEV) , hog cholera virus (HCV) , foot and mouse disease virus(FMDV) , porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) in IIF test. The results suggested that these three MAbs were probably only specific to the N protein of PRRSV. In SDS-PAGE and Western blot analysis, all results showed that 5A5 reacted with N protein, but not reacted with Sf9. As the result, these 3 hybridoma cells were able to secret antibodies stably, which reacted with PRRSV specifically in HF test. All these results indicated that three monoclonal antibodies based IIF or immunohistochemical (IHC) staining could be potentially used for the diagnosis of PRRS and detection of PRRSV.In order to develop an IIF to diagnose PRRS, 3 3-week-old pigs were experimentally infected with PRRSV ATCC VR-2332 strain and 2 pigs with both PRRSV ATCC VR-2332 and PCV2. Three age-matched pigs were served as uninoculated controls. Serum was collected before and after inoculation. Two infected pigs and one control pig were necropsied at one, two and three weeks postinfection (WPI) , respectively. Samples (serum, lung, tonsil, spleen, inguinal lymph node) were collected at the same time and tested for evidence of PRRSV infection by observing of microscopic lesions. The PRRS V-specific antibody response was monitored with HerdChek IDEXX Kit. It was certificated in serum antibody monitoring that all serum samples were negative before inoculation and became positive after 2 WPI. Slight pathology was observed in lung and results of IIF attempts on lung showed that all samples were positive except for control pigs. All results were positive for evidence of PRRSV infection. In IIF test, PRRSV was monitored by these 3 MAbs and detected in the lung and spleen. Summarizedly, these 3 MAbs could be used to detect PRRSV in clinical cases.7 PRRS-suspected pigs were detected and the results showed that only 2 pigs from Shuyang were PRRSV positive in IIF test while the others were negative. Virus isolation (VI) was preceded and the presence of PRRSV in cell lysates was confirmed by IIF test. Results of VI attempts on lung tissue showed that cytopathic effect (CPE) was observed at the fourth passage and the fluorescence occurred in cytoplasm of infected Marc-145 cells. All results demonstrated that these 3 MAbs against N protein of PRRSV could be used to detect PRRSV in clinic. Otherwise, two isolates from Shuyang could be used in PRRSV molecular epidemic study, and their subtype would be identified soon.