Preparation And Characterization Of Monoclonal Antibodies Against Porcine Parvovirus VP2 Protein

Porcine Parvovirus(PUCcine Parvovirus,PPV)is a major pathogen of porcine reproductive disorders.The disease is causing great economic losses to the pig industry and widespread all over the world.The genome-encoded VP2 protein is the major capsid protein of the virus and carries the main antigenic determinants,which determine the main biological properties of the virus,such as tissue tropism,pathogenicity and antigenicity.In this study,the recombinant baculovirus Ac NPV-VP2 stored in the laboratory was inoculated into the insect cells Sf9,after 36 hours,the virus was collected and analyzed by SDS-PAGE,and then analyzed by Western blot.The results showed that the protein was highly expressed successfully.The VP2 protein was purified by resin and gelatinization,and subjected to Western blot analysis using monoclonal antibody of anti-His tag protein.The results showed that the purified protein could be used in the follow-up study.6 weeks-aged BALB/c mice were immunized subcutaneously using the purified VP2 protein,after three primary immunizations and one enhanced immunity,the cells were screened by indirect ELISA using cell fusion technology and were expandly cultured by limited dilution method,more than 3 times of cell subclone,finally three hybridoma cells secreting anti-VP2 protein antibody stably were obtained,respectively named 1B3,1E9 and 5F8.The subtypes of 3 monoclonal antibodies were all identified as Ig G1.The titer of hybridoma cell culture supernatant was 1: 800-1000,and the hybridoma cells were injected intraperitoneally into BALB/c mice which had been injected with paraffin for one week,and the obtained ascites titer was 1: 32 000-64 000.To further identify the immunoreactivity of three monoclonal antibodies,PPV was inoculated with ST cells and the virus was collected as an immunogen to detect the immunogenicity of monoclonal antibodies.Western blot analysis showed that all the 3 monoclonal antibodies could identify the PPV whole virus and the recombinant VP2 protein.The results of indirect immunofluorescence showed that all the monoclonal antibodies reacted with PPV whole virus.Specific tests showed that the 3 monoclonal antibodies only specifically reacted with PPV but not with TEGV and PEDV.Neither culturing in vitro for 3 months nor cryopreservation could affect the stability of antibody secretion of the 3 hybridoma cells.In this study,three monoclonal antibodies anti porcine parvovirus structural protein VP2 were successfully prepared,and they all had high reactogenicity and specificity,which laid a theoretical basis for establishing effective PPV-specific serological detection method subsequently,and alsoprovided the material basis for the study of PPV receptor and infection mechanism.