Screening And Identification Of B Cell Epitope Of P30 Protein For African Swine Fever Virus

African swine fever(ASF)is a highly pathogenic infectious disease caused by African swine fever virus(ASFV)and charactered by high fever and multiple organ hemorrhage and enlargement.ASF poses a serious threat to animal health,food safety,national economy and environment.ASFV is a large double-stranded DNA arbovirus with a complex structure,and it is the only member of the family Asfarviridae.Due to the lack of in-depth understanding of ASFV protective immunity,there is still no effective vaccine available.Among the structural proteins that compose the virion of ASFV,p30 is one of the important antigen structural proteins.It is is expressed in the early stage of viral infection of host cells.Because of its high immunogenicity,p30 is often used as a serological candidate protein for ASF diagnosis and monitoring.Preparation of p30 monoclonal antibody(Monoclonal antibody,m Ab)and further screening of p30 protein antigen epitope have great significance for in-depth study of the biological function of p30 protein,and will also help to develop detection methods for ASFV.In this study,the recombinant lentiviral particles were constructed by p LVX-IRES-Zs Green1-p30/p TRIP-CMV-Puro-p30,ps PAX-2 and p MD2.G plasmids,then transfected into H22/HEK293T cells.H22 cell strain and HEK293T cell strain were screened and identified by indirect immunofluorescence assay(IFA)and Western Blot.These two cell strains could stably express ASFV p30 protein.Then,mice were immunized with p LVX-IRES-Zs Green1-p30-H22 cells.After four times of animal immunizations,five monoclonal cell lines were screened by using cell fusion technology and p TRIP-CMV-Puro-p30-HEK293T cells as the detection antigen.Immunoperoxidase monolayer assay(IPMA)was used to screen five monoclonal cell strains.Western Blot verification showed that the monoclonal antibodies generated by these cell lines could recognize the p30 protein expressed in the eukaryotic expression system.Finally,according to the overlapping peptide method,13 segments of p30peptides(P1-P13)were synthesized,each overlapping 4 amino acid(Amino acid,aa).Peptide-enzyme-linked immunosorbent assay(Peptide-ELISA)was used to detect the response of peptides to ASFV-positive pig serum and post-immunized mouse serum.The results showed that peptides P2(16-35 aa),P5(61-80 aa),P6(76-95 aa),P10(136-155 aa),P12(166-185 aa)and P13(181-201 aa)were dominant peptides in p30 protein.Peptide-ELISA was used to detect the reaction between peptides and p30 m Ab.The results showed that P2,P5 and P6 could be recognized by 9C7 m Ab,and P6 could be recognized by 2E7 m Ab,and P12 could be recognized by 10D7 m Ab.In order to further identify the B cell epitope region recognized by2E7 and 10D7 m Abs,P6 and P12 were truncated and reacted with monoclonal antibody.The results showed that 10D7 m Ab recognized P12-2 and P12-3polypeptides,and located their epitopes in the region ¹⁷¹YGTPLKEEEK¹⁸⁰to¹⁷⁶KEEEKEVVRL¹⁸⁵,which was the first reported B cell epitope,which was the first reported B cell epitope for p30 protein.In this study,p30-specific monoclonal antibody was prepared using H22 cells stably expressing p30 protein as immunogen,and the linear B cell epitopes of p30protein were identified.The above results are helpful to understand the interaction between antigen and antibody,and have great significance for the detection and diagnosis of ASFV.