Establishment Of Indirect ELISA Antibody Detection Method Based On African Swine Fever Virus Multi-epitopes Fusion Protein

African swine fever is a highly fatal infectious disease caused by African swine fever virus in pigs.Proteins such as p30,p54 and p72 encoded by ASFV are often used as serological diagnostic targets to evaluate the changes of antibody levels in different stages or degrees of ASF.The complexity of proteins encoded by ASFV brings challenges and opportunities to the development of new methods for serological diagnosis of ASF.In-depth study of ASF serological diagnostic targets would be helpful to explore its infection detection,pathogenic mechanism and immune system response.In this study,we collected or predicted the B cell epitopes of p30,p72,CD2 V,MGF360-505 R proteins of ASFV Chinese epidemic strains,the antigenic epitopes tandem recombinant proteins was designed,then the MGF360-505R-CD2 v protein was selected for study of Indirect ELISA method for detection of Ig G antibody.The specific results are as follows:1.Analysis of antigenic epitopes(regions)of serological diagnostic targets(p30,p72,CD2 V and MGF360-505R)of ASFV Chinese epidemic strains and design of epitopes tandem proteins.Bioinformatics analysis showed that the sequences of p30,p72 and CD2 V proteins of ASFV epidemic strains in China were highly conserved,ASFV p30 protein deletes the first 8 amino acid(MDFINIS)in Wuhan1 and Wuhan2 strains,the difference of amino acid at position 194 in SY18 strain(K difference is I),and more than amino acid(SFFLTYI)at position 195 to 201,the homology of other sequence regions can still reach 100%.Based on the collected or predicted B cell epitopes and solubility prediction analysis of p30,p72 and other proteins,the ASFV p30 epitopes tandem recombinant protein(8 epitopes),p72 epitopes tandem recombinant protein(8 epitopes),p30-p72 epitopes tandem recombinant protein(12epitopes)and MGF360-505R-CD2 v epitopes tandem recombinant protein(19 epitopes)were designed respectively.The identification of the protein epitopes of ASFV is helpful to better understand the pathology and host immune response of the virus.2.Expression,purification and identification of ASFV epitopes tandem recombinant proteins(p30,p72,p30-p72,MGF360-505R-CD2v).The epitopes tandem recombinant proteins of p ET21-p30,p ET28a-p72,p ET28a-p30-p72 and p ET28a-MGF360-505R-CD2 v were expressed in prokaryotic expression system(E.coli),The results showed that the expression of p30 and MGF360-505R-CD2 v epitopes tandem proteins was higher,that of p30-p72 epitopes tandem protein was good,and that of p72 epitopes tandem protein was poor.The epitopes tandem recombinant proteins of p30,p30-p72 and MGF360-505R-CD2 v were soluble to a certain extent.The inclusion body of MGF360-505R-CD2 v tandem epitope protein was successfully purified.The MGF360-505R-CD2 v tandem epitope protein was expressed in eukaryotic system(HEK-293 cells).The expression level was good and the nickel column was successfully purified.Western blotting assay further identified that ASF serum antibody could recognize MGF360-505R-CD2 v epitopes tandem proteins expressed in prokaryotic system(Escherichia coli)and eukaryotic system(HEK-293 cells).3.Establishment of indirect ELISA method using MGF360-505R-CD2 v epitopes tandem protein.The MGF360-505R-CD2 v epitopes tandem recombinant protein expressed in prokaryotic system(E.coli)was selected as coating antigen,and an ELISA method for detection of Ig G antibody was established.The systems and reaction conditions were determined systematically : the optimum coating concentration of antigen protein is 4ng/μL,detection of serum using 100 times dilution multiple,the dilution ratio of enzyme-labeled antibody was 1:7000,the suitable reaction time for the detection of serum antibody is 30 min,the most suitable reaction time for TMB substrate is 5min.The results of further experiments show that the ELISA method has good specificity,sensitivity and repeatability.The preliminary application results of clinical samples showed that the comprehensive coincidence rate with ID-VET kit was 92.93%(92/99),the establishment of this indirect ELISA method has certain reference value for ASF detection,serological detection based on conservative epitopes is expected to provide a wider range of diagnosis.