Surface Display And Antigenicity Analysis Of African Swine Fever Virus P30 Protein

African swine fever(ASF)is a globally prevalent swine infectious disease caused by African swine fever virus(ASFV).ASFV-infected pigs display a variety of clinical symptoms ranging from acute to chronic,and may die within days.Since there is currently no vaccine or treatment for ASF,the disease has spread uncontrollably to many countries in Europe,South America,North America,Asia,and more.China reported the first case of ASF in August 2018,and it has been prevalent since then.As of July 16,2021,192 outbreaks occurred nationwide,and more than 1 million pigs were culled,causing huge losses and serious threats to China’s and even the global pig industry.At present,it is urgent to develop rapid and efficient diagnostic reagents for ASF to control the ASF epidemic,develop safe and effective vaccines to prevent ASFV invasion,and provide guarantee for domestic pig breeding.In this study,ASFV P30 protein was displayed on the surface of baculovirus virion envelope or virus-infected cells to study its immunogenicity.Using Bac-to-Bac transfer vector plasmid pFPG as the skeleton,the fusion expression frame gp64SP-Fp30-gp64TMD was inserted into the downstream of baculovirus membrane fusion protein gene gp64 promoter,which contained the signal peptide sequence of gp64 gene and fused p30 gene with FLAG label.Meanwhile,the transmembrane region and intracellular region of gp64 gene were fused downstream.The expression frame is expected to transfer the P30 protein to the membrane surface by expressing GP64 signal peptide and to anchor to the membrane or viral envelope via transmembrane and intracellular domains.The recombinant plasmid was named PFPG-GP64-FP30.After PCR,double enzyme digestion and sequencing verification,the constructed recombinant plasmid was transformed into DH10Bac for transposition,and then the eukaryotic expression vector containing P30 gene was obtained by resistance and blue-white spot screening.Sf9 cells were transfected to obtain recombinant baculovirus BACmid-GP64-Fp30.The recombinant baculovirus expression product was 23.6kDa by SDS-PAGE and Western-blot analysis,which was consistent with the expected size.In addition,immunofluorescence showed that P30 could anchor on the surface of Sf9 cells.After Sf9 cells were infected with recombinant virus for 96 h,lysed cells were collected as immunogens for animal immunization test.Serum of immunized mice was collected for indirect ELISA and Western-blot test.The positive serum titer of immunized mice was 1:12,800.The results showed that the target protein P30 antigen exhibited on the surface had strong immunogenicity,and Western-Blot and indirect ELISA showed that it could induce strong specific antibodies.The excellent immunogenicity displayed on the surface of P30 protein lays a good foundation for the development of highly sensitive diagnostic kits for ASFV and the study of ASF vaccine.