Generation And Characterization Of Monoclonal Antibodies Against P30 And P54 Proteins Of African Swine Fever Virus

African Swine Fever(ASF)is an acute,febrile,highly contagious and deadly viral haemorrhagic swine disease caused by the African Swine Fever Virus(ASFV).It mainly affects domestic pigs and wild boars with morbidity and mortality rates up to 100%,and is transmitted by an arthropod vector,soft ticks of the genus Ornithodoros.The disease was first discovered in Kenya in 1921,spread to countries outside Africa after two transcontinental transmissions in 1957 and 2007,and was introduced to China in 2018.Due to the complexity of the ASFV genome and limited knowledge of ASFV infection and immune evasion mechanisms,currently the vaccine is still under development.So the prevention and control of ASFV mainly relies on early detection and early culling.Therefore,selecting virus proteins with strong antigenicity and immunogenicity to generate their specific antibodies,and consequently establishing a rapid and accurate detection and diagnosis method is critical for the prevention and control of ASFV.ASFV p30 and p54 are strong immunogenic structural proteins expressed in the early stage of ASFV infection,and are important targets for early detection of ASFV infection.In this study,we successfully generated 2 strains of p30 monoclonal antibodies and 1 strain of p54 monoclonal antibody,and checked their specificity,which laid foundation for developing early diagnosis of ASFV infection and studying biological functions of p30 and p54 proteins.The study specifically includes the following two aspects:1.Prokaryotic expression and purification of p30 and p54 recombinant proteinsIn this study,the nucleotide sequences of p30 and p54 encoding genes CP204L and E183L of several ASFV strains isolated in China were compared with the ASFV genotype? Georgia 2007 strain(ASFV Georgia 2007/1),and then the fragments with 100%homology and high antigenicity were selected.PCR primers of p30 and p54 were designed based on the nucleotide sequences of CP204L and E183L genes of ASFV Georgia 2007/1 strain,respectively.The p30 and p54 target fragments were amplified using plasmids expressing CP204L or E183L of ASFV Georgia 2007/1 strain,pcDNA3-2×Flag-CP204L or pcDNA3-2×Flag-E183L,as a template.Amplified fragments were analyzed by nucleic acid electrophoresis,followed by gel purification,double enzyme digestion and ligation to the pET-28a vector.The positive clones were screened and verified by sequencing.After transformation the successfully constructed prokaryotic expression plasmids pET-28a-p30 and pET-28a-p54 into E.coli Rosetta,p30 and p54 expression were induced by IPTG.After SDS-PAGE electrophoresis,the results showed that p30 and p54 proteins were mainly expressed in inclusion bodies.Finally,the p30 and p54 proteins were purified by His-tagged protein purification nickel column and the AKTA protein purification system,and p30 and p54 recombinant proteins with high purity were obtained.2.Generation of monoclonal antibodies against p30 and p54 proteinsIn this study,purified p30 and p54 proteins were used as immunogens to immunize 4?6-week old BALB/c mice.After three times of immunization,through cell fusion,limiting dilution,and indirect ELISA,2 strains of hybridomas with stable secretion of anti-p30 monoclonal antibodies and 1 strain with stable secretion of anti-p54 monoclonal antibody were obtained.Monoclonal antibodies were amplified by injecting hybridoma cells into the abdominal cavity of mice and were characterized by subtype identification,Western-blot,indirect immunofluorescence assay and blocking ELISA.The results showed that the two strains of p30 and one strain of p54 monoclonal antibodies obtained in this study could specifically identify corresponding viral proteins expressed in ASFV-infected cells and have certain blocking effect,which laid an important foundation for future research on the early detection of ASFV and the biological functions of ASFV p30 and p54 proteins.