Preparation Of Monoclonal Antibody Against Prrsv Nsp9Gene And Establishment Of Block-ELISA Method

Porcine reproductive and respiratory syndrome (PRRS) is one of the viralinfectious diseases. It has brought huge economic losses to the world’s pig industry.At present, the OIE list PRRSV as class B infectious diseases. Although the liveattenuated vaccine play an important role in controling of PRRSV, and it willcontinue to use in the future, it can not be ignored that live attenuated vaccine existsthe potential of returning virulence. So PRRSV-negative farms should careful use thelive attenuated vaccine.Commercialization vaccines of PRRS divide into inactivated and theattenuated vaccines. Both vaccines are able to reduce the PRRS incidence, but couldnot prevent the occurrence of virulent infection and persistent infection. There aresome differences in PRRSV isolates, such as different genotypes, biologicalcharacteristics, antigenic, genetic characteristics. Therefore, use of single PRRSVstrains prepared vaccine is not fully protect the vaccinated pigs against heterologousinfection. This brought great difficulties to the PRRS vaccine′s development.Especially the outbreaks of H-PRRS in recent years make people question the qualityof conventional PRRS vaccine. The key to improve the immune effects isdevelopment of new PRRS vaccine, or different immune pathways use differentvaccine, or use different vaccine, or search new immunoadjuvant.At present, the serological detection methods are the important methods ofmonitoring PRRSV infection, but the traditional serological detection methods can notaccurately distinguish between infected pigs and vaccinated pigs.The PRRSV Nsp9gene which code RNA-dependent RNA polymerase (RdRp) participate in the processof viral replication. In view of the inactivated virus can not copy, the body can notproduce Nsp9antibodies.So it provides a theoretical basis that use Nsp9protein asdiagnostic antigen to distinguish the field virus and vaccine virus.The the diagnosis ofPRRSV strains are not mature, these methods are laborious and unsuitable for clinicaltesting. So it is urgent need a diagnostic method for simple, rapid, specific inclinically test.In this project, we expressed PRRSV Nsp9protein, prepared anti-Nsp9monoclonal antibody, and established a blocking ELISA method of distinguishbetween the field and attenuated virus. In this study, we artificial expressed of PRRSV Nsp9protein which had goodimmunogenicity, and successfully prepared the anti-Nsp9protein monoclonalantibody2D6,3C11,4E6. Western-blot analysis, immunofluorescence andimmunohistochemistry show that the three monoclonal antibodies had good activity.We select2D6to develop a blocking ELISA method for detection of PRRSV. Afteroptimization of conditions, change the method to distinguish the inactivated immuneand live viral infection.170clinical sera test show that blocking ELISA detection rateof68.8percent, while the commercial kit detection rate of85.9%, indicating that theblocking ELISA method is more sensitive, the correlation of the two methods is97.8%.