Screening And Identification Of Immunogenic ASFV-encoded Membrane Proteins

African swine fever(ASF)is a highly pathogenic infectious disease caused by African swine fever virus(ASFV),the clinical symptoms of which include fever,respiratory and nervous system dysfunction,and internal organ disease with extensive hemorrhage.However,there is no effective commercial vaccine that can be used for preventing or treating until now.Once found,the only available approach is to be killed on a large scale in farms.Therefore,finding the protein with good immunogenicity encoded by ASFV is of great significance for investigating the virus pathogenicity and developing related detection kits.Using TMHMM analysis software,the 169 protein sequences encoded by ASFV genome were analyzed,and it was found that the 35 protein encoded by ASFV genome has one or more transmembrane regions.Using WB and IFA methods,it was found that E199 L,KP117R,E183 L,B169L,DP148 R,E146L,EP84 R,EP402R,H108 R have obvious feature of cell membrane localization.In this study,169 possible genes of ASFV genome were synthesized based on the published open reading frames(ORFs)of the ASFV HLJ/18 isolate genome,and cloned into pCAGGS-Flag vector.Subsequently,169 eukaryotic expression plasmids were successfully constructed.After these ASFV encoded proteins are expressed in HEK293 T cells,the challenged pig serum(ASF positive serum)was used to identify by Western blotting and indirect immunofluorescence and obtained 9 ASFV proteins reacted well with ASF positive sera as follows: p12(O61R),p54(E183L),P30(CP204L),pK145 R,pK205R,pCP312 R,p17(D117L),pA137 R,pA104R,of which p12(O61R)),p17(D117L),and p54(E183L)are membrane proteins with transmembrane regions,suggesting that these ASFV encoded proteins may have good immunogenicity.According to the hydrophobicity of p17 and the previous analysis,the 61-117 aa of p17 was intercepted to synthesize small peptides,as the coating antigen.And then,the optimal antigen coating concentration and the best dilution of the tested serum,the best blocking solution and blocking time were screened.Besides,we selected the working time of the tested serum,incubation conditions of rabbit anti-pig IgG enzyme-labeled antibody,and color development time,and thus,an indirect ELISA detection method for ASFV p17 was initially established.In addition,based on our previous results,we found that unlike the Belin and BAV71 strains,the virulence-related protein,pDP148 R,encoded by the ASFV HLJ/18 strain genome contains only 237 amino acids.The C-terminus of the protein may contains a nuclear-localized sequence,but the over-expressed full-length DP148 R located on the cell membrane of HEK293 T and can cause cell death.We purified the pDP148 R protein through a prokaryotic expression system.After immunization of the mice,spleen cells were fused with myeloma cells to obtain the hybridoma cells and successfully prepared monoclonal antibody against DP148 R protein.To summarize,in this study,we found that 35 proteins encoded by ASFV genome are membraneproteins with transmembrane regions and confirmed that 3 of them have good immunogenicity.At the same time,we preliminarily established the indirect ELISA diagnostic method of p17 and prepared the monoclonal antibody of p DP148 R successfully.Our study will lay a solid foundation for the research of the biological functions of ASFV important functional membrane proteins and the development of ASF detection methods,and will also provide scientific support for the prevention and control of ASF.