Preparation Of Monoclonal Antibody Against A151R Protein Of African Swine Fever Virus And Establishment Of Indirect ELISA Detection Method

African swine fever(ASF)is a highly contagious disease that causes severe hemorrhaging.The disease’s pathogen is the African swine fever virus(ASFV).Because the disease has the characteristics of rapid transmission,short course of disease,and extremely high mortality,it has caused substantial economic losses and harm to China’s pig industry.At present,the ELISA detection method for ASFV has been widely used.The ELISA method for viral protein detection is simple and fast,does not require expensive equipment,and can quickly detect pathogens on the farm site,which is of great significance for clinical diagnosis,prevention and control of ASF.However,the cost of imported ELISA detection kits is high,and domestic ELISA detection kits are low in price but low in sensitivity.Therefore,there is a need to research new target proteins for detection.The ASFV genome ranges from 170 to 193 kb and can encode nearly 200 viral proteins.Among them,A151 R is a non-structural protein associated with ASFV replication and assembly.It is expressed during both the early and late stages of viral infection,exhibits strong immunogenicity,and serves as an effective target for the development of ASF diagnostic tools.1.Bioinformatics analysis and soluble expression of African swine fever virus A151 R proteinIn this study,the gene sequence of A151 R protein from ASFV China/2018/Anhui XCGQ strain was analyzed by bioinformatics software,and the codon was optimized and synthesized.The prokaryotic expression vector of A151 R protein was constructed,and soluble expression of recombinant protein was promoted by coexpression and purification of chaperone protein.The purified recombinant protein A151 R was obtained by affinity chromatography and gel filtration.This protein exhibited strong immunogenicity,with a reaction titer of 1:12627 against ASFV standard positive serum.2.Preliminary establishment of indirect ELISA for detection of African swine fever virusUsing purified A151 R protein as the coating antigen,an indirect ELISA method for ASFV detection was established.By optimizing the antigen coating concentration,serum dilution,incubation time,secondary antibody dilution,and incubation time,as well as the development time,an indirect ELISA method based on the A151 R protein was developed to detect ASFV antibodies.This method showed good specificity,with no cross-reactivity observed with other common pig viral sera in clinical samples.However,the agreement rate with commercial test kits was relatively low.Testing 60 serum samples revealed that among the samples that tested positive with the commercial test kit(20 samples),19 samples tested positive using the indirect ELISA method.Additionally,among the samples(40 samples)that tested negative with the commercial test kit,more than half(28 samples)tested positive using the indirect ELISA method,resulting in an agreement rate of 51.67%.Further examination and analysis are required to determine the specific reasons for this discrepancy.3.Preparation of monoclonal antibody against African swine fever virus A151 R proteinThree monoclonal antibodies were generated by immunizing mice with the prokaryotically expressed and purified A151 R protein.Western blot and immunofluorescence assay(IFA)results demonstrated that all three monoclonal antibodies specifically reacted with the antigen expressed in eukaryotic cells.These antibodies can be utilized for Western blot detection and cellular-level analysis of the A151 R protein,providing valuable materials for further investigations into the protein’s function.