| A virus strain was isolated from a serum sample which obtained from the Swine herd suffered a reproductive syndrome in Fujian Province. By futher analysis and identification, the isolate was proved to be reproductive and respiratory syndrome virus (PRRSV), and the isolated virus was named as PRRSV-FJ0601 strain.The antigen of PRRSV FZ strain was purified by differential centrifugation and discontinuous sucrose density gradient centrifugation. The Balb/c mices were immunized with purified viral antigen. And the spleen cells from immunized mice were fused with Sp2/0 myeloma cells to prepare the PRRSV specific antibodies secreted by hybridoma cell line. After screening the hybridomas with indirect enzyme linked immunosorbent assay (ELISA), and three times subcloning, three hybridoma cell lines were developed, which could steadily secret specific monoclonal antibodies(McAbs) against PRRSV-FZ. These McAbs were designated as McAb-A61,McAb-B47 and McAb-C65, respectively. Their biological characteristics were analysed. And the McAb-A61-FITC conjugate was prepared and used for develping a direct immuno-fluorescent assay(DFA) for detecting PRRSV. The results were summarized as follows:1. McAb-FJ0601 strain isolation and identification. A virus strain was isolated from serum samples of the diseased piglets, which could induce the CPE in the Marc-145 monolayer in 72hrs post-inoculation, but didn't cause any CPE in PK-15 and Vero cells. The neutralization assay proved that the virulence of the isolated virus could be inhibited by PRRSV antiserum; immunofluorescent test showed the virus could be recognized by McAbs against PRRSV; RT-PCR with the PRRSV specific primer and the genomic RNA produced predicted 443bp DNA fragment. All the above results demonstrated the virus was PRRSV. The isolated virus was named as PRRSV-FJ0601.2.The isotyping analysis results showed that the McAb-A61 and McAb-C65 belonged to IgG1 and McAb-B47 belonged to IgG3; All the three McAbs could react with strains of PRRSV-FZ, PRRSV-2332 and PRRSV-FJ-1 by ELISA, but no one had cross-reactivity with Marc-145, Porcine parvovirus(PPV) and Porcine circovirus Type 2(PCV2); Their ELISA titers of the supernatant and ascites fluid were ranged from 26～28 and 105～106 respectively. Western blot analysis confirmed that the three McAbs could recognise 26 KD virus structure protein(PRRSV GP5). IFA test indicated that McAb-A61 and McAb-B47 could react with antigens of PRRSV-FZ and PRRSV-2332 , whereas McAb-C65 could recognize all of three PRRSV strains. The phenomenon suggested that the McAbs binding site of the PRRSV-FJ-1 could have a different distribution on virus by compared with PRRSV-FZ and PRRSV-2332 stains. Therefore, under treating with organic solvent, the conformation change was happened on the epitope of PRRSV-FJ-1 and caused the disability to bind with the McAb-A61 and McAb-B47. -3.The ascites fluid of McAb-A61 was purified and labeled with fluorescein siothibcyanate(FITC). A direct fluorescent antibody (DFA) test using the conjugate as probe was developed for detecting the PRRSV antigen. The results showed the FITC conjugated McAb-A61 only reacted with PRRSV infected cells, did not have any reaction with PPV, PCV2 and normal Marc-145 cell.The successfully preparation of the McAbs to PRRSV and McAb-A61-FITC conjugate provided a useful tool to study the structure proteins of the PRRSVand the rapid diagnosis of the PRRSV antigen. |
