Development And Characterization Of Monoclonal Antibody Against GP5Protein Of Porcine Reproduction And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV belongs to the family Arteriviridae, and has two distinct genotypes:Europe type and American type. GP5protein plays an important role in virus infection and is reported to contain neutralizing epitopes. Thus the production of anti-GP5mAb is of great value in immunodection of the virus and treatment of the disease.In this study, GP5was successfully constructed and expressed in E.coli BL21(DE3). A series of conditions such as IPTG concentration, induction time, induction temperature and so on were optimized. The most appropriate condition was in37℃,0.6mM IPTG, induction time is6h. GP5protein mainly existed as inclusion body. Recombinant GP5was denatured with8M urea and broken by ultrasound. GP5protein was purified by nickel affinity column. SDS-PAGE analysis showed that degeneration PRRSV-GP5protein expression successfully with its molecular weight is16kDa.Western-blot analysis showed that the truncated GP5protein showed high specific reaction to the PRRSV positive serum.PRRSV serum antibodies indirect ELISA method was established based on recombination PRRSV-GP5as detecting antigen. By detecting the mice positive serum and pig positive serum as a competitive antigen, the effective positive serum was selected and used as inhibit antibody to establish blocking ELISA method of screening mAb. Different time of infected cells, fixed agent, the fixed time, an antioxidant and two resistant to dilute concentrations and reaction time was optimized to set up indirect immunofluorescence (IFA) and immunoperoxidase monolayer assary (IPMA).The recombination GP5protein was used to immune BALB/c mice according to the conventional method. The ELISA titer of the third immune was1:6400. Three days before cell fusion, BALB/c mice were super vaccine by tail intravenous with GP5protein.Inhibit ELISA and IFA were applied to screen monoclonal antibody (mAb). Immune BALB/c mice the spleen cells were fused with NSO cells according to the conventional method.Firstly,58positive cloning(OD>0.45) were screened using recombinant GP5protein as detect antigen with indirect ELISA methods. Then the positive clone was screened by inhibit ELISA with the pig PRRSV positive serum. Three strong positive cloned cells were three times limiting dilution. Three strains of stably secreted the mAb of PRRSV GP5protein was set up. Three hybridomas producing were obtained which could screen monoclonal antibody (mAb) specific for the GP5protein after screening by blocking ELISA and ELISA. They were named4B7,2C5and2E4.The ELISA titers of cell supernatant and ascites of4B7were1:800and1:51200respectively. Ig subclass of the mAbs were determined as IgG1. All of the mAbs were shown to react specifically with the Marc-145cells infected with PRRSV BJ-4strain in IFA and IPMA, indicating they could recognize antigen epitope of the native viral protein of PRRSV.These functional mAbs provide effective technology for PRRSV immune recognition and rapid diagnosis.