Expression And Monoclonal Antibody Preparation Of The P54Recombinant Protein Of African Swine Fever Virus

African swine fever (African swine fever, ASF) is a highly contagious disease of domestic pigs caused by African swine fever virus (African swine fever virus, ASFV) with mortality up to100%. The disease was first reported in Africa and has transmitted recently to our neighboring countries such as Georgia and Russia, which threatens the swine industry of China. Since there is no vaccine against the disease, establishment of quick accurate diagnostic methods is critical to prevention of the disease entry and dissemination.The p54protein is one of the major structural proteins of ASFV, which has been used as the antigen for serodiagnosis of ASFV. In this study, we amplified ASFV p54gene by PCR and inserted into prokaryotic expression vector pET-30a (+). The recombinant plasmid pET30a-P54was transformed into BLR (DE3) E. coli and expression of the His-p54fusion protein was induced with IPTG SDS-PAGE showed that the His-p54fusion protein was expressed in a soluble form with an expected molecular weight of21kDa. By using nickel-affinity chromatography column, the fusion protein was purified to a high purity.BALB/c mice were immunized with the purified His-p54fusion protein and their spleen cells were fused with mouse myeloma cells SP2/0. The positive cell clones were screened by indirect ELISA using purified His-p54protein as the antigen. After3cycles of sub-cloning, two positive cell clones were obtained. Western-blotting and indirect fluorescent assay showed that the antibodies reacted positively with p54protein. The two hybridoma cell lines were injected into peritoneal of mice and the ascites monoclonal antibodies were harvested with ELISA titers of216and213, respectively.