Preparation and Identification of Monoclonal Antibody against gE Protein of Porcine Pseudorabies Virus
In order to develop an immunodiagnostic reagent for porcine pseudorabies virus(PRV),BALB/c mice were immunized with the recombinant gE protein expressed in HEK293F cells.The spleen cells of immunized mice and SP2/0 myeloma cells were fused to generate hybridoma cells.Indirect ELISA and immunoperoxidase monolayer assay(IPMA)were used to screen positive hybridoma cells so as to prepare and identify monoclonal antibodies against the gE protein of PRV.The outcomes demonstrated that two hybridoma cell strains,named 2B5 and 8F7,respectively,which steadily secreted monoclonal antibody against the gE protein were developed.The ELISA titers of ascites were all 1∶1 000 000.IgG1 was the heavy chain and Kappa was the light chain,according to the monoclonal antibody isotype assay.The specificity assay revealed that the two monoclonal antibodies only reacted with PRV and not with other viruses.SDS-PAGE results showed that the purified monoclonal antibodies had high-purity specific bands at about 50 ku and 25 ku.The two monoclonal antibody strains could react specifically with 293T cells transfected with gE plasmid according to indirect immunofluorescence assay(IFA)detection.Western blot showed that the specific protein band appearing in the culture supernatant of two hybridoma cells was about 120 ku,indicating that the two monoclonal antibodies could specifically recognize PRV.To summarize,this study successfully prepared two strains of monoclonal antibodies against gE protein,exhibiting excellent specificity and high titer,which provided crucial biological materials required for the subsequent development of diagnostic kits.
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