Preparation Of Monoclonal Antibody Against African Swine Fever Virus P54 Protein And Its Epitope Identification

African swine fever(ASF)is a viral hemorrhagic disease caused by the African swine fever virus(ASFV)that causes a persistent chronic infection without clinical symptoms in African wild pigs and ticks.It can cause different clinical syndromes in domestic pigs and Eurasian wild boar with mortality up to 100%.Despite the limited host range and lack of zoonotic potential,the socio-economic impact of ASFV is so high that the disease must be reported to the World Organization for Animal Health(OIE).Control methods for ASF rely on strict hygiene measures,and although ASF has been extensively studied,there is still a lack of effective vaccines or antiviral strategies,and many questions remain about the molecular mechanisms of the infection cycle.The p54 protein,one of the important structural proteins of ASFV,is encoded by E183 L gene and located in the inner capsule of the virus.When the virus infects cells,the microtubule motor complex transports the virus by directly binding to the cytoplasmic dynamin light chain.The p54 protein is also the main antigenic protein of ASFV.The p54 antibody in serum has an inhibitory effect on virus adhesion of porcine macrophages,with an inhibitory rate of about 60%.Therefore,the p54 protein can be used as one of the target proteins of ASFV for the preparation of monoclonal antibody,and can also be used as the main material for serological diagnosis.In this study,prokaryotic codon optimization was performed on the extracellular region sequence of p54 protein encoded by ASFV E183 L gene,and 6×His tag was added to synthesize it into the prokaryotic expression vector p ET-30 a plasmid.The recombinant p54 protein was expressed by IPTG induction using the Escherichia coli expression system.The recombinant p54 protein was purified by Ni column affinity chromatography and immunized BALB/c female mice.After three times of immune stimulation,mouse serum was used to detect the serum titer by indirect ELISA method.Mice with the highest serum titer of p54 antibody were selected for shock immunization.Then spleen was extracted and ground to prepare single cell suspension for cell fusion,which was used to prepare monoclonal antibody against ASFV p54 protein.Hybridoma cells secreting monoclonal antibody to p54 were screened by indirect ELISA method.Six stable monoclonal cell lines were obtained by subcloning three times.The cell line was treated with colchicine for chromosome number analysis.The monoclonal antibody subtypes were identified using kits,and the 6 monoclonal cell lines were prepared by ascites.The p54 monoclonal antibody in ascites was crude extracted by saturated ammonium sulfate method,and the affinity activity of the purified monoclonal antibody was detected by ELISA.The recombinant p54 protein was expressed eukaryotically by p CAGGS-Flag-p54,and the specificity of monoclonal antibody to p54 was determined by Western blot and indirect immunofluorescence assay(IFA).Cross-reactivity test was performed to identify the reaction of six monoclonal antibodies with other swine pathogens.According to the predicted secondary structure of p54 protein,the region where the linear epitopes of monoclonal antibody were located was studied by Western blot by truncating the prokaryotic expression of p54 peptide.Recombinant p54 protein was successfully obtained,which could be recognized by His monoclonal antibody and ASFV positive serum.The recombinant p54 protein was coated with 1.25 μg/m L and the serum was diluted 1:1,600 to establish an indirect ELISA method with good specificity and simple operation.Six hybridoma cell lines with stable secretion of p54 monoclonal antibody were successfully prepared and named as 28G12-1,31G7-1,31G7-2,35F10-1,35F10-2,38D3-1 and the antibody subtypes were identified as Ig G2 a,Ig G2 a,Ig G2 a,Ig G1,Ig G1 and Ig G1,respectively.The average number of chromosomes in the six monoclonal cells was 102,which was about the total number of chromosomes in the parent cells.The titers of purified p54 monoclonal antibodies were 1:124,400-1:409,600.Western blot and IFA analysis showed that the six monoclonal antibodies were specific.The cross-reactivity test showed that the monoclonal antibodies did not react specifically with other pathogens,indicating that the six monoclonal antibodies of p54 had good specificity.The epitope region of p54 monoclonal antibodies recognized by truncation method,was identified as 127-146 aa of the C-terminal and showed on the predicted tertiary structure of p54 protein.In conclusion,this study successfully obtained ASFV p54 protein with good immunogenicity.Six high affinity monoclonal antibodies were prepared,which could react specifically with p54 protein.The region of the epitopes recognized by the six monoclonal antibodies was preliminically identified,which laid a foundation for the study of the function of p54 protein,the infection mechanism of ASFV and the research of peptide vaccine.