Screening Of Dominant Antigen Of African Classical Swine Fever Virus And Establishment Of Indirect ELISA For PE184L Protein

African swine fever(ASF)is an acute,hot,contact and highly lethal infectious disease caused by African swine fever virus(ASFV).Since the first case of African swine fever in China was reported in 2018,the epidemic has spread rapidly.Due to the lack of effective vaccine,it has a serious impact on the pig industry.Early diagnosis is the key to ASF prevention and control.The market also urgently needs fast and accurate early diagnosis methods for ASF.ASFV encodes more than 200 proteins.In addition to the main structural proteins,the function of non structural proteins is unknown,which also hinders the in-depth study of ASFV.In order to further understand the structural and functional characteristics of ASFV protein,in order to screen the potential dominant antigen of ASFV,establish a rapid and accurate ASFV detection method,and lay a foundation for the effective prevention and control of ASFV.Firstly,the dominant B-cell epitopes and structural characteristics of eight ASFV nonstructural proteins(p H124 R,p C129 R,p K145 R,p O174 L,p E184 L,p I329 L,p MGF-360-14 L and p NP419L)were evaluated and analyzed by bioinformatics method.Secondly,eight ASFV recombinant expression plasmid vectors were constructed by synthesizing the target gene,and the ASFV recombinant protein was expressed in prokaryotic.Combined with ELISA,the eight recombinant proteins were reacted with ASFV positive serum to analyze the immunogenicity.After screening the dominant ASFV recombinant protein,immunized mice to analyze its immunogenicity.Finally,the chessboard titration method was used to determine the best reaction conditions for screening the dominant protein antigen,establish an indirect ELISA detection method,and evaluate it from the aspects of sensitivity,stability,specificity and coincidence rate.The results showed that:(1)ASFV p H124 R,p C129 R,p K145 R and p O174 L were stable hydrophilic proteins,and p E184 L,p MGF-360-14 L,p I329 L and p NP419 L were unstable hydrophilic proteins.The amino acid sequences of 8 proteins are mainly composed of α Spiral and β Folding composition,sequence composition of p NP419 L β Corner and irregular curl account for a high proportion.p I329 L is a transmembrane protein with signal peptide structure.p E184 L and p NP419 L have four dominant B-cell antigen epitopes respectively,p H124 R,p K145 R and p I329 L have three dominant B-cell antigen epitopes respectively,and p H129 R,p O174 L and p MGF-360-14 L have two dominant B-cell antigen epitopes respectively.(2)Eight recombinant plasmids of ASFV proteins(p ET-28a-H124 R,C129R,K145 R,O174L,E184 L,MGF-360-14 L,I329L,NP419L)were constructed.Six soluble proteins(p H124 R,p C129 R,p K145 R,p O174 L,p E184 L,p NP419L)and two inclusion body expressed egg whites(p MGF-360-14 L,p I329L)were successfully purified,and the protein purity was more than 90%.The results of ASFV positive serum reactivity showed that the OD values of eight ASFV proteins were between 0.522-1.095,and the OD value of p E184 L protein was1.095,indicating that p E184 L protein had good reactivity.After immunizing mice,the titer of anti p E184 L protein serum was 1:64000,indicating that it has good immunogenicity.(3)The optimum reaction conditions of ASFV p E184 L protein indirect ELISA were as follows: the best coating solution was carbonate buffer(p H: 9.6),the best coating time was 1h,the best coating concentration of p E184 L antigen was 1.25 ng / UL,the best blocking solution and blocking time were 5% skimmed milk,blocked at 37 ℃ for 1 h,the best dilution of blood sample was 1:200,incubated for 15 min,The optimal dilution of enzyme labeled secondary antibody was 1:10000,incubation for 15 min,color development for 20 min,and cut off value was 0.123.The coefficient of variation of intra batch and inter batch repeated experiments of the established ASFV p E184 L protein indirect ELISA method was less than10%,and there was no cross reaction with the positive sera of HCV,PRRSV,GBV,PCV2 and other viruses.The results showed that among 161 clinical samples,83 were positive and 78 were negative for ASFV p E184 L protein indirect ELSIA.The total coincidence rate was96.27%.In conclusion,the characteristics of 8 ASFV nonstructural proteins were comprehensively analyzed by bioinformatics methods.By constructing the prokaryotic expression vector of ASFV recombinant protein,8 ASFV nonstructural proteins were successfully prepared.Combined with the results of immunogenicity analysis of bioinformatics and reactivity analysis with ASFV positive serum,p E184 L protein was selected as the dominant antigen,and the serum titer test results of immunized mice showed that,ASFV p E184 L protein has good immunogenicity.Based on the above comprehensive evaluation results of p E184 L protein,an indirect ELISA method for ASFV p E184 L protein was established.This method has good sensitivity,specificity and stability,and has a good coincidence rate compared with the commercial ASFV ELISA kit.This study provides a basis for the screening of potential ASFV dominant antigens,the establishment of early and rapid diagnosis methods of ASF and the effective prevention and control of ASF.