Identification Of B Cell Epitopes On P30 Protein Of African Swine Fever Virus

African swine fever(ASF)is a highly contagious and lethal viral disease in domestic and wild swine,affecting all breeds and ages of pigs.ASF is caused by African swine fever virus(ASFV),which is extremely complex in structure.Among the structural proteins that compose the virion of ASFV,p30 is highly immunogenic and is produced during early stage of ASFV infection.In the absence of ASF vaccines,the development of monoclonal antibody against ASFV P30 protein and the screening and identification of antigen epitopes are helpful for the prevention and control of ASF.In this study,a molecular chaperone(pGro7/pET28a-p30)co-expression system was constructed using the prokaryotic expression system and the induction conditions were optimized.His Trap Q HP and His Trap FF were used to purify the target protein.The optimal induction conditions were 21?,0.1 mmol/L IPTG and 0.05 mg/mL L-arabinose final concentration for 8 h.After purification by His Trap Q HP affinity column and His Trap FF,sodium dodecyl sulfate-poly-acrylamide gel electrophoresis(SDS-PAGE)results showed that the purity of the target protein could reach more than 80%.Western Blot results showed that the target protein could react specifically with anti-His monoclonal antibody.The mice were immunized with the purified protein,and the serum titer of the mice reached 1:1.024×105.Monoclonal antibodies(mAbs)against ASFV p30 protein were prepared by cell fusion.Three mAbs,named 7D2,8C8 and 2F6,were obtained.The titer of 7D2 reached 1:1.024×106,the affinity constant of 7D2 reached 1.77×109 L/mol.The titer of 8C8 reached 1:2.048×106,the affinity constant reached of 8C81.9×1010 L/mol.The titer of 2F6 reached 1:5.12×105,the affinity constant of 2F6 reached 8.93×109 L/molThe full-length amino acids sequence of ASFV p30 protein was screened using three mAbs as the target molecules.Two new linear B cell epitopes of ASFV p30 protein were identified after two rounds of peptide-ELISA and Dot-ELISA.The epitope sequences were 26VFHAGSLYNW35 and 1MDFILNISMKMEVIFKTDLR20 respectively.Conservation analysis indicated that epitope 1MDFILNISMKMEVIFKTDLR20 and 26VFHAGSLYNW35 were highly conserved among the different strains of ASFV.The epitope peptide were displayed in the simulated three-dimensional structure of p30 protein,in which 26VFHAGSLYNW35 was partially exposed on the surface of p30 protein structure,and 1MDFIL NISMK MEVIF KTDLR20 was completely exposed on the surface of p30 protein structure.Both they can react with ASFV positive pig serum.In our study,we obtained the recombinant p30 protein with high purity and good immunogenicity,prepared three mAbs.Two new linear B cell epitopes of ASFV P30 protein were screened:26VFHAGSLYNW35,1MDFIL NISMK MEVIF KTDLR20,which were the first reported B cell epitopes for p30 protein.These findings lay the foundation for the enrichment of ASFV p30 protein epitopes.