Identification Of B Cell Epitopes Of The Intracellular Domain Of African Swine Fever Virus CD2v And Verification Of Immune Activity

African Swine Fever(ASF)is a highly contagious disease caused by African Swine Fever Virus(ASFV)that infects pigs,resulting in high morbidity and mortality rates and significant economic losses in the global pig industry.ASFV is a linear double-stranded DNA virus with a genome size ranging from 170 to 190 kbp that encodes 151 to 167 proteins.CD2 v is an outer membrane protein encoded by the EP402 R gene,consisting of a signal peptide,transmembrane region,and two immunoglobulin-like domains,and is commonly used for preparing immunoprotective antigens.Recent studies have reported multiple B-cell linear epitopes in the extracellular domain of CD2 v,some of which can induce effective immune responses in a mouse model.However,B-cell linear epitopes of the CD2 v intracellular domain protein(CD2v-IR)and its immune activity are unknown.In this study,we expressed CD2v-IR in Escherichia coli,identified one B-cell linear epitope using monoclonal antibodies prepared from this protein,and validated its immune activity in a mouse model.1.Prokaryotic expression of ASFV CD2v-IR and eukaryotic expression of CD2v-Full and CD2v-IRBased on the published ASFV/HLJ strain EP402 R gene sequence in Gen Bank,the CD2v-IR sequence was codon-optimized and cloned into the pET28 b prokaryotic expression vector,named pET28b-CD2v-IR.The plasmid was transformed into BL21(DE3)competent cells and induced by IPTG to express the CD2v-IR.After affinity chromatography and gel filtration purification,the CD2v-IR with a purity of over90% was obtained.Western Blot results showed that the obtained product was consistent with expectations,which provides a good foundation for the preparation of monoclonal antibodies.Primer was designed to connect the CD2 v full-length protein(CD2v-Full)and CD2v-IR sequence to the pFast Bac HTC vector to obtain the recombinant transfer plasmids pFast Bac HTC-CD2v-Full and pFast Bac HTC-CD2v-IR.Both plasmids were transformed into DH10 Bac competent cells and screened by blue-white selection to obtain the recombinant bacmid plasmids Bacmid-CD2v-Full and Bacmid-CD2v-IR.These two recombinant baculovirus vectors were transfected into Sf9 cells,cultured and continuously passaged for three generations to obtain protein samples.Western Blot confirmed that the constructed baculovirus successfully expressed the CD2 v full-length and intracellular domain proteins,providing a material basis for the subsequent detection of CD2 v monoclonal antibodies.2.Preparation of monoclonal antibodies against ASFV CD2v-IR and screening of its linear B cell epitopesASFV CD2v-IR expressed and purified in a prokaryotic system was used to immunize 6-8 week-old female Balb/c mice.Spleen cells from the mouse with the highest serum titer were fused with SP2/0 cells,and a hybridoma cell line 1F3 stably secreting monoclonal antibodies was obtained through two rounds of subcloning by indirect ELISA.Western Blot and IFA results showed that the antibody specifically reacted with the eukaryotic expressed CD2v-Full,CD2v-IR,and inactivated PAM cell samples infected with ASFV.Western Blot confirmed that the recognized epitope by1F3 was located at 264-270 aa,and further Dot Blot and ELISA assays determined264-270 aa as the minimal epitope recognized by 1F3.3.Verification of the immune effect of ASFV CD2v-IR and its B-cell linear epitopeASFV CD2v-IR was expressed in prokaryotic cells and purified for immunization of 20 female Balb/c mice aged 6-8 weeks.The mice were randomly divided into four groups: PBS,BSA,CD2v-IR,and epitope peptide(peptide conjugated with BSA),with five mice in each group.Immunization was performed on days 0 and 28,and tail blood was collected every 7 days after immunization.On day14 after the second immunization,blood was collected from the mice’s eye sockets to obtain serum,and splenocytes were collected from the spleen.ELISA was used to detect antibodies,flow cytometry was used to analyze lymphocyte subtypes,and ELISA mouse cytokine detection kits were used to detect cytokines.The results showed that both CD2v-IR and epitope peptide could induce effective humoral and cellular immune responses.In summary,CD2v-IR was expressed in a prokaryotic expression system and purified to immunize mice,and a monoclonal antibody 1F3 was screened against CD2v-IR.Then a linear epitope recognized by 1F3 located at 264-270 aa.Using CD2v-IR and its epitope as immunogens,the immunogenicity of the epitope peptide was validated in a mouse model,providing a material basis for the development of an ASFV epitope vaccine.