Production And Characterization Of Monoclonal Antibodies Against African Swine Fever Virus CD2v Protein

African swine fever(ASF)caused by African swine fever virus(ASFV)is a hemorrhagic and contagious disease.ASF causes up to 100% mortality rate in domestic pigs,which is becoming an economic impact and serious threat to the piggery worldwide.CD2 v is the key pathogenic factor and the protective antigen of ASFV,which is one of the target proteins of ASF vaccine design and immunoassay.In this study,the recombinant CD2 v proteins were expressed in HEK293 F cells to achieve the secretory expression and glycosylation modification.Using purified recombinant CD2 v protein as immunogen,the monoclonal antibodies(m Abs)with high titer and high affinity activity were screened and identified.1.Eukaryotic expression and purification of ASFV CD2 v proteinThe CD2 v extracellular region sequence of ASFV SY18 strain was inserted into loop2 of the Norovirus(No V)P particle,and assembled into nanoparticles,which was named by CD2 v protein nanoparticle(P-CD2v).The CD2 v extracellular region sequence was cloned into the vector in the same method,named by monomer CD2v(CD2v).After eukaryotic expression,the recombinant CD2 v proteins were purified by His Trap Excel column.The results showed that the molecular weight of P-CD2 v protein was about 110 k Da,and the molecular weight of monomer CD2 v protein was about 75 k Da.The particle size results showed that P-CD2 v protein could spontaneously assemble into a particle size of 19.9 nm,while monomer CD2 v protein was a particle size of 2.6 nm.2.Preparation and screening of m Abs against ASFV CD2 v proteinThe mice were immunized with the same CD2 v content,using purified recombinant CD2 v proteins as an immunogen.Measured by indirect ELISA and compared to the monomer CD2 v protein,P-CD2 v protein induced higher antibody titers of immunized mice.In the meanwhile,P-CD2 v protein produced m Abs with higher titer,and m Ab 10A3 had the highest titer of 1:2048000.Then,m Ab 10A3 was used to produce CD2 v antibody column with ideal coupling efficiency,which provided a more convenient and rapid method for the purification of CD2 v protein without label.3.Identification of m Abs against ASFV CD2 v proteinMAbs induced by P-CD2 v protein had higher antibody titer and affinity activity.Six m Abs induced by P-CD2 v protein were identified by dot-blot,IFA and IPMA.The results of Dot-blot and IFA showed that the screened m Abs could specifically bind the recombinant CD2 v protein.IPMA results showed that the six m Abs could react with the formaldehyde fixed cells infected with ASFV HLJ/18 isolates,and could recognize natural virus.The blocking ELISA results based on m Abs showed that ASFV positive serum could block the reaction of six m Abs with the recombinant protein,among which,m Ab 2E9 had the highest blocking inhibition rate(84%)to ASFV positive serum,indicating that the m Ab 2E9 had good specificity and sensitivity.It can be used to establish serological detection method of ASF.4.Identification of antigen epitopes of ASFV CD2 v proteinWestern blot results showed that the six m Abs induced by P-CD2 v protein reacted specifically with the denature CD2 v protein,which could be used for linear epitope recognition.According to the predicted epitopes and Ig superfamily(Ig SF)related domains,the CD2 v extracellular region was truncated,and the truncated plasmid was transfected into HEK293 T cells to be used for the identification of antigen epitopes.In this study,the linear epitope with the location of amino acids 28 th to 51 st of CD2 v extracellular domain sequence was identified,and the location was showed on the simulated CD2 v protein structure.In conclusion,the CD2 v protein nanoparticle with high immunogenicity was generated,m Abs with high titer and high affinity activity were screened,and a precise location of antigen epitope on CD2 v was determined.These results may provide powerful help for etiological and serological detection methods of ASFV.