Development And Characterization Of Monoclonal Antibody Against The Critical Loop Structure Of African Swine Fever Virus P72 Protein

African swine fever(ASF)is a highly contagious and 100% lethal swine viral disease that has existed for over 100 years and has caused severe losses to the global pig industry.African swine fever virus(ASFV)is the causative agent of ASF,and P72 is the key pathogenic factor of ASFV and a highly conserved key antigen.The four prominent Loop structures present on the surface of the P72 protein presumably contain crucial protective antigenic epitopes.Therefore,it is imperative to develop monoclonal antibodies specific to these Loop structures for effective ASFV diagnosis and vaccine development.In this study,the four crucial loops(ER1-4)of ASFV P72 protein were embedded onto hepatitis B virus core particles(HBc)to self-assembling virus-like particles(VLPs),which maintain the natural conformation of its Loop structure and enhance their immunogenicity to generate high-titer murine P72 monoclonal antibodies(m Abs)and precisely locate key epitopes in the P72 sequence.1.Prokaryotic expression and purification of the structural protein ER1-4 of African swine fever virus P72 LoopER1-4 of ASFV P72 were embedded in the Major immunodominant region(MIC)of HBc to self-assemble into VLPs named VLP ER1,VLP ER2,VLP ER3,and VLP ER4,respectively.After recombinant VLPs were expressed in prokaryotic,they underwent purification through salting(saturated ammonium sulfate precipitation)and Core 700.The results showed that the molecular weight size of VLPs ER1-4 was about 25 KDa,and the four VLPs were successfully expressed by self-assembly as identified by transmission electron microscopy.2.Preparation and screening of monoclonal antibody to P72 protein of African swine fever virusThe purified recombinant VLPs ER1-4 protein mixed with Freund’s adjuvant was used as an immunogen to immunize mice,and monoclonal antibodies were obtained through hybridoma cell preparation technique,limited dilution method subcloning,and indirect ELISA assay screening.A total of 10 hybridoma cell lines were obtained with the highest ascites potency up to 8 000:204 800.3.Identification of monoclonal antibodies to the P72 protein of African swine fever virusRecombinant plasmids p CAGGS-P72 and p CAGGS-B602 L were constructed for eukaryotic expression,which co-transfect into HEK-293 F cells to express the full-length P72 protein.Pretty reactivity and specificity of P72 expression were confirmed by SDS-PAGE and Western blot analysis.Ten monoclonal antibodies,induced by recombinant VLPs ER1-4 protein,were identified through Western blot,Dot-blot,IFA,and IPMA.All isolates exhibited binding activity and were able to recognize the natural ASFV.The results of blocking ELISA experiments based on monoclonal antibodies showed that all ASFV-positive sera could impede the reaction of six monoclonal antibodies with full-length P72 protein.Among these,monoclonal antibody 4G8 displayed the highest blocking inhibition rate of 84% for ASFV-positive sera,indicating high specificity and sensitivity.These findings may serve as a reference for establishing effective measures against ASFV.4.Identification of antigenic epitopes of African swine fever virus P72 proteinWestern blot results showed that ten monoclonal antibodies induced by VLP ER2 and VLP ER4 proteins were reactive with denatured P72 protein and could be used for linear epitope recognition.ER2 and ER4 proteins were truncated based on the predicted antigenic epitopes from IEDB and Pymol.The resulting sequences were synthesized by Bio and coupled with BSA for epitope identification.Amino acids 250-274,282-299,and 507-517 located in the P72 sequence were successfully identified as linear epitopes according to Dot-blot,demonstrating highly conserved.In summary,in this study,VLPs for the highly immunogenic ASFV P72 Loop structural proteins ER1-4 were successfully constructed,and ten high-titer monoclonal antibodies to the ER region of ASFV P72 protein were prepared and characterized for their specificity.The precise location of the linear epitope of P72 was clarified,which can provide a powerful help for the etiological and serological detection studies of ASFV.