Preparation And Identification Of Antibodies Against Ber Pathway Proteins Encoded By African Swine Fever Virus

African Swine Fever(ASF)is an acute,febrile,highly infectious and highly fatal viral disease of pigs caused by the African Swine Fever Virus(ASFV).It mainly affects domestic pigs and wild boars with a morbidity and mortality rate up to 100%.ASFV naturally infects host monocytes/macrophages.As immune cells,macrophages can stimulate active oxygen reaction to damage the virus genome to inhibit virus replication.In contrast,the virus activates a series of proteins that repair damage to the genome,such as Base Excision Repair(BER)that oxidation or hydrolysis-induced base damages of viral DNA.Interestingly,several ASFV BER pathway proteins were identified,including O174L-encoded DNA polymerase,D345L-encoded exonuclease and NP419L-encoded DNA ligase.Exonuclease excises redundant bases in the long patch repair pathway.DNA polymerase pO174 L acts on AP sites to fill in the missing base,DNA ligase connects breakage ends of the DNA strands.In addition,DNA ligase pNP419 L can induce a high level of antibody titer in ASFV-infected pigs,indicating that it is a potential diagnostic target.The purpose of this study is to prepare polyclonal antibodies against pO174 L and pD345 L and monoclonal antibodies against pNP419 L,which can provide reagents for further studying of the biological functions of these proteins and developing diagnostic kits.The research is consists of the following two parts:1.Preparation and identification of polyclonal antibodies against African swine fever virus pD345 L and pO174 L proteinsPCR primers of D345L(173-346 amino acids)and O174 L genes were designed according to the SY-18 strain sequence published on NCBI.The target genes were amplified using pcDNA3-2×Flag-D345 L and pcDNA3-2×Flag-O174 L as templates,and analyzed by nucleic acid gel electrophoresis.After enzyme digestion,the target fragments were inserted into pET-28 a or pET-32 a,respectively.The ligation products were transformed into DH5a competent cells and then extract plasmids which were analyzed by double enzyme digestion and sequencing.Then positive recombinant plasmids pET-28a-D345 L and pET-32a-O174 L were transformed into Rosetta competent cells and induced by IPTG to express pD345 L and pO147 L recombinant proteins.SDS-PAGE electrophoresis analysis showed that pD345 L expressed in the inclusion body and pO174 L expressed in the supernatant and inclusion body.After purified by nickel column,we got pD345 L and pO174 L proteins with high purities.The mice were immunized with purified recombinant proteins emulsified with adjuvant.After finishing the immunization procedure,mice blood was collected to isolate polyclonal antisera.Finally,Western-blot results showed that both polyclonal sera could recognize the pD345 L and pO174 L proteins expressed in pcDNA3-2×Flag-D345 L and pcDNA3-2×Flag-O174 L transfected 293 T cells,or in ASFV-infected PAM cells well.2.Preparation and identification of monoclonal antibodies against African Swine Fever Virus pNP419 L proteinThe same as above,PCR primers of NP419 L gene were designed,and the eukaryotic plasmid pcDNA3-2×Flag-NP419 L was used as a template to amplify the target gene.The positive pET-28a-NP419 L plasmid was constructed by restriction enzyme digestion,ligation,transformation,enzyme digestion analysis and sequencing.After the plasmid was transformed into Rosetta competent cells and induced by IPTG,the expression of pNP419 L protein was identified by SDS-PAGE,and the results showed that pNP419 L protein was expressed in the inclusion body.After purified with a nickel column,the recombinant protein with a high purity was obtained.Mice were immunized with emulsified proteins.After two times of immunizations,serum antibody titers were detected by ELISA,and the mouse with a high antibody titer was selected to make boost immunization.One week later,mouse spleen cells were fused with myeloma cells SP2/0.Three monoclonal cells(8G1,9D1,10F8)secreting pNP419 L antibody were screened by limited dilution,subcloning and indirect ELISA analysis.Western-blot results showed that three monoclonal antibodies could recognize the pNP419 L proteins expressed in pcDNA3-2×Flag-NP419 L transfected cells or ASFV-infected cells.Examination of the antibody subtype showed that the heavy chain of three monoclonal antibodies were Ig G type.The pNP419 L protein and monoclonal antibodies were used for indirect ELISA and blocking ELISA detection.The results showed that indirect ELISA method could detect antibodies in clinical positive serum samples,while blocking ELISA method showed poor blocking effect.In summary,polyclonal antibodies that can specifically recognize pO174 L and pD345 L proteins expressed in virus-infected cells and three monoclonal cell lines secreting pNP419 L specific antibodies were obtained in this study.In addition,pNP419 L protein and its monoclonal antibodies were used for indirect ELISA and blocking ELISA to establish detection methods.This study laid a foundation for further research,optimization and development of an indirect ELISA detection kit.