Establishment And Application Of An Indirect ELISA Method Based On Recombinant Protein Of ASFV Antigenic Epitope Peptides

African Swine Fever(ASF)is an acute,highly contagious,and severe infectious disease in pigs caused by African Swine Fever Virus(ASFV)with mortality rate up to 100%.There is no safe and effective vaccines and drugs available for ASF prevention and therapy.Accurate monitoring is critical to prevent ASF outbreak.Recent studies have found that low-virulence gene deletion strains are prevalent in the past two years inducing latent infection.False-negative results may occur in ASFV-related pathogen detection.Therefore,it is necessary to develop sensitive,specific,and low-cost serological detection methods for accurate ASF diagnosis.ASFV p72,p54,and p30 proteins are commonly used as targets for serological diagnostic method development.However,there are still deficiencies including unstable expression and high cost for protein purification.Here,we established a novel,rapid,and accurate ASF serologic detection method based on stable expressed recombinant proteins as follows:1.Truncated p72 protein expression and monoclonal antibody preparationTo screen for more p72 antigenic epitopes for recombinant protein design.The truncated p72 gene(1269-1923 bp)was inserted into the p ET28 b vector to express the protein,and monoclonal antibodies were prepared by immunizing BALB/c mice.After three subclones screening,western blot and IFA results indicated that a specific Ig G1 subtype monoclonal antibody against ASFV p72 is successfully generated.2.Verification of B cell epitopes of p72,p54,and p30 proteins.We first used p72 monoclonal antibody to recognize a B cell epitope477SDQNPHQHRDWHKFGH492.To obtain more B cell epitopes of p72,p54,and p30 proteins,we utilized bioinformatics to predict and got six the B cell epitope information of,p54,and p30 proteins,which specifically react with ASF positive serum,respectively.3.Recombinant protein designation and ASF indirect ELISA method establishmentASF antigenic epitope recombinant protein was designed by the Alpha Fold2 software.After that,the gene of recombinant protein was synthesized and inserted into the p ET30 a vector for large-scale expression.Western blot indicated that the recombinant protein can specifically react with ASF positive serum and p72 monoclonal antibody、p54 and p30 polyclonal antibodies,respectively.ASF indirect ELISA method was established based on the recombinant protein,which has no specific reaction with other important pig viral disease serum.Meanwhile,it shows a sensitivity of detecting dilutions of ASF standard positive serum up to 1:6,400.The clinical sample detection results showed a high conformity rate of 98% with the commercial competition ELISA kit.In summary,we identified six antigenic epitope peptides and generated a recombinant protein.Using this recombinant protein,we established a ASF indirect ELISA detection method,which provides material basis for ASF diagnosis and control.