Preparation Of Monoclonal Antibody To CD2v Of ASFV And A Novel Epitope Identification Of P30

African swine fever（ASF）is a highly contagious disease caused by African swine fever virus（ASFV）infecting pigs,which is the only known DNA arboviruses with double-stranded DNA.It is characterized clinically by high fever,systemic hemorrhage and high mortality.ASF is classified by the OIE as a legally reported animal disease and is a class I animal disease in China.There is no recognized effective vaccine or treatment method up to now.The CD2v protein encoded by the ASFV EP204R gene is a late expressed protein,a transmembrane structural protein that has been found to be present in the capsid of the virus and is involved in intercellular adhesion,virulence enhancement and immune regulation.2 immunoglobulin-like structural domains in the extracellular region have high homology with mammalian CD2 and are associated with the in vivo transmission of the virus.the ASFV CP204L gene encodes The structural protein p30,located in the inner membrane of the viral envelope,is present in large numbers in the early stages of viral infection and induces the production of neutralising antibodies in the body,while the antigenic epitopes are widely used in vaccine development and diagnostic reagent development.Understanding the interaction between anti-ASFVp30 antibodies and epitopes will allow further research into the diagnosis,detection and prevention and control of ASF.In this study,the extracellular region of CD2v protein was expressed by a prokaryotic vector expression system,and four anti-CD2v monoclonal antibodies were developed by hybridoma technology,with a view to providing corresponding biological materials for the prevention,control and diagnosis of the disease.The study also identified a novel antigenic epitope of African swine fever.1.Cloning and expression of the extracellular region gene of African swine fever virus CD2vIn this study,a pair of amplification primers specifically targeting the extracellular region of CD2v protein（1aa～206aa）was synthesized according to the sequence of CD2v protein,and the target gene was amplified by PCR and cloned into the vector pET-32a.pET-32a-CD2v was transformed into BL21 receptor cells,and expression was induced by adding IPTG.SDSPAGE analysis of the supernatant and precipitate showed that the protein was mainly present in the form of inclusion bodies.Western Blot with His-tagged monoclonal antibody and ASFVpositive pig serum as primary antibody showed that the molecular weight of the recombinant protein was about 38 kDa.The recombinant protein was purified and the purified protein was analyzed by SDS-PAGE with a target band at around 38kDa and no other spurious bands,which provided material for the subsequent development of monoclonal antibody against CD2v protein.2.Development of monoclonal antibodies against CD2v extracellular region of African swine fever virusIn this study,the purified CD2v extracellular region recombinant protein was used as the immunogen,and BALB/c mice aged 6-8 weeks were immunized.The serum potency of the mice was determined after three immunizations,and the mice with higher potency were intensively immunized,and their spleen cells were fused with myeloma cells.MA-104 cells infected with ASFV were fixed,and the supernatants of hybridoma cells were screened by IFA,and the positive cells were subcloned to obtain four cell lines that stably secreted anti-ASFV CD2v antibodies,named ASFV-CD2v-2B6,ASFV-CD2v-4B6,ASFV-CD2v-4C2 and ASFVCD2v-4D4,respectively.The subclass identification showed that all four monoclonal antibodies were IgM subclasses/Kappa chains and all reacted specifically with ASFV.The IFA potency of ASFV-CD2v-2B6 and ASFV-CD2v-4B6 monoclonal antibodies was determined to be 1:6400,and that of ASFV-CD2v-4C2 and ASFV-CD2v-4D4 monoclonal antibodies was 1:1600.3.Identification of a novel antigenic epitopes of p30The monoclonal antibody ASFV-p30-3F9,which has a high IFA potency,was used as the target.pET-32a was used as the expression vector to truncate the p30 protein,and the reaction of different truncated proteins with the monoclonal antibody ASFV-p30-3F9 was verified by Western-blot.The results showed that the shortest antigenic epitope recognized by the monoclonal antibody ASFV-p30-3F9 was 178 EEKEVRLM 186.25 domestic and foreign ASFV strains from GenBank were selected for the conservativeness analysis,and the epitope was found to be highly conserved among different strains,which laid the foundation for the study of p30 protein function and monitoring of ASFV.