Screening And Application Of Dominant Antigen Of African Swine Fever Virus

African swine Fever(ASF)is an acute and highly fatal infectious disease for pigs caused by African swine Fever virus(ASFV).Since 2018,ASF first broke out in Shenyang,China,and then spread rapidly to the whole country,causing serious economic losses to the pig industry.ASF has been classified into Class I animal disease.At present,there is no available effective vaccine and drug.With the extension of ASFV epidemic in China,the main pathogenesis of ASF changed from large-scale outbreak to sporadic occurrence,and the attenuated ASFV strains became more and more.The prevalence of attenuated strains require sensitive serological antibody detection.Due to the large number of proteins encoded by ASFV,the viral antigen reaction is complex.This study proposed by prokaryotic expression is mainly structural protein and high quantity of protein expression,to positive ASFV swine serum screening the advantage of the virus antigen,and advantages of antigen protein as envelope antigen detection ASFV antibody indirect ELISA method,provides the technical support for ASFV diagnosis and at the same time also provide related vaccine research and development of evaluation methods.In this study,16 highly expressed proteins and 32 main structural proteins were identified as screening antigens based on the proteomic analysis of African swine fever virus particles and infected cells.According to the genome sequence of the popular GII ASFV strain in China,the gene sequence of the analysis antigen was determined,and the antigenicity of ASFV-related protein was analyzed by Protean software,meanwhile,the transmembrane region was analyzed by TMHMM-2.0 software,so as to select the dominant antigen fragment,and the gene sequence was optimized by the Escherichia coli codon optimization system.Primer 5.0 software was used to design primers for PCR amplification,and the amplified target fragment was connected to the p Cold-I vector.Identification and sequencing results showed that p Cold-I-p30 and p54 were successfully constructed.46 viral gene sequences including KP177R,K205R,EP153R,CD2V,A104R and p72 were synthesized and cloned into p Cold-I vector.In this study,fifty recombinant plasmids were successfully constructed and induced.The proteins expressed in the precipitation after ultrasound were purified by inclusion body method.Others that expressed in supernatant after ultrasound was purified by Ni+affinity chromatography.Finally,36 target proteins were successfully purified by SDS-PAGE analysis.36 purified target proteins were used as coated antigens,and the negative/positive sera under different antibody levels were compared in parallel by indirect ELISA.The OD₄₅₀nm reading value was performed,and the results were determined according to the ratio of positive pore(P)/negative pore(N),and the dominant antigen was screened.Among them,3 antigens showed strong positive reaction in all positive serums,17 antigens showed strong positive reaction in partial positive serums,3 antigens showed weak positive reaction in partial positive serums,13 antigens showed negative reaction in all positive serums,and none of them showed positive reaction in negative serums.After evaluation,p30 was selected as the best dominant antigen.These results indicated that48 ASFV gene prokaryotic expression plasmids were successfully constructed and 36 target proteins were successfully purified.p30 was identified as the best dominant antigen.Based on the above results,we established an indirect ELISA method for detecting African swine fever virus p30 protein.First,the purified ASFV p30 protein was coated with an enzyme plate.The optimal conditions were determined by square titration,OD₄₅₀nm was used to read the value,and the results were judged according to the ratio of positive(P)/negative(N)Wells.The experimental results showed that the optimal antigen coating concentration was 20 ng/well,and the optimal serum dilution was 1:40.Further optimization,when coated with carbonate coating solution and sealed with 5%skim milk at 37?for 1h,the best effect was obtained.The optimal coating temperature and time were 37?1h,the optimal dilution ratio of HRP-conjugate secondary antibody was 1:10000,the optimal reaction time of serum and secondary antibody was 1h,and the optimal color developing temperature and time was room temperature(21?-25?)10min.Based on the optimal conditions of the above experiments,24 ASFV standard negative serum samples with known background were detected.According to the principle of statistics,the determination domain of indirect ELISA antibody detection method was determined:negative value should be?0.244,positive value should be?0.294,and the two values were suspicious.In specificity test,there was no cross reaction between this method and standard positive serum of PRRSV,SIV,PRV,PEDV,CSFV and PDCOV,indicating that this method has good specificity.In the sensitivity test,the method showed positive reaction when the positive serum was diluted to1:1,280,indicating that the method had good sensitivity.The indirect ELISA antibody assay was used to detect 180 clinical samples from the field and compared with the commercial ASF assay kit.The results showed that the coincidence rate was 98.3%.In conclusion,in this study,prokaryotic expression plasmids of 48 ASFV genes were successfully constructed by prokaryotic expression system and 36 target proteins were successfully purified.After screening,the dominant antigens of ASFV infection were identified as p30,p54 and KP177R,among which p30 protein was the best dominant antigen.The indirect ELISA method was constructed based on ASFV p30 protein,which had good specificity and sensitivity.