Construction And Immunological Characteristics Of Baculovirus Vectors Expressing African Swine Fever Virus Proteins

African swine fever(ASF)is an acute,intense and highly contagious infectious disease caused by African swine fever virus(ASFV)infecting domestic pigs and wild boars,with a fatality rate of up to 100%.African swine fever caused enormous economic loss to Chinese pig breeding industry.However,so far,there is no effective means of prevention and control.One important reason is that the function of the protein encoded by ASFV is largely unknown.In the reality of biosafety prevention and control,the early warning of African swine fever risk depends on accurate,efficient and low-cost detection methods.Therefore,in this study,recombinant baculovirus expression vectors of 6 important genes in ASFV were constructed,and indirect ELISA detection method was established to provide an effective tool for the development of new ASFV vaccines and accurate diagnosis.In this study,six important antigens in ASFV gene(A151R,B438 L,K205R,A104 R,B602L,MGF505-5R)were selected for the construction of recombinant baculovirus vector.At the same time,the above genes were linked into two baculovirus shuttle vectors in series to obtain the following results: p Fast Bac1-CMV-EGFP-A151R-B438L-K205RA104R-HA,p Fast Bac1-CMV-EGFP-B602L-MGF505-5R-HA.After transposition of all the recombinant shuttle vectors,Bacmid was extracted.After PCR verification was correct,Sf9 cells were transfected,and after obvious lesions were observed,the cells were passed through,and strong fluorescence was detected by indirect immunofluorescence method(IFA).The recombinant baculovirus was successfully expressed by Western Blot,which detected the target bands with the corresponding size.In this study,recombinant B602 L protein purified in laboratory was used as antigen coated enzyme plate,and an indirect ELISA method was established for the detection of recombinant B602 L protein.The final determination procedure was as follows: coated antigen 1μg/m L,coated at 4 ℃ overnight after 2 h at room temperature.3 % skim milk sealed for 3 h;The sera to be tested were diluted 1:80 and incubated at 37 ℃ for 120 min.The 1:10000 diluent enzyme-labeled secondary antibody was incubated at 37 ℃ for 90 min.When TMB was treated at 37 ℃ for 10 min,the color development was terminated by2 MH2SO4.The results of repeatability test showed that the coefficient of variation within batches was 3.80%-8.80%,and the coefficient of variation between batches was4.72%-7.82%,both of which were less than 10%.An indirect ELISA assay based on B602 L and a commercial assay kit(ID.Vet)were used to detect 60 clinical serum samples at the same time,showing an overall compliance rate of 95%(57/60).In summary,6 baculovirus expression vectors and 2 tandem baculovirus expression vectors were successfully constructed in this study,and corresponding proteins were successfully expressed by IFA and WB verification,with good immunogenicity.In this study,an indirect ELISA assay based on B602 L was established for the detection of ASFV antibody,with good specificity,sensitivity and repeatability.