Preparation of Monoclonal Antibody Against African Swine Fever Virus p54 Protein and Identification of Its Epitope

The aim of this study was to obtain soluble African swine fever virus (ASFV) p54 protein and anti-p54 monoclonal antibodies (mAb), along with the identification of their recognized epitopes, so as to lay a foundation for the study of the structure and function of p54 protein and the development of serological diagnostic reagents.

To prepare mAb against ASFV p54 protein, the prokaryotic recombinant expression plasmid pET-21a-E183L was constructed and transformed into E.coli BL21 (DE3) cells. The recombinant protein p54 was purified by Ni-affinity chromatography and gel filtration, five weeks old BALB/c mice were immunized with the purified p54 recombinant protein. The immunization protocol involved three rounds at two-week intervals, starting with an emulsion of the antigen and an equal volume of Freund’s complete adjuvant, followed by the same antigen emulsion with an equal volume of Freund’s incomplete adjuvant for the subsequent immunization. After three immunizations, the blood of mice was collected, and the serum antibody titers were detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titers were selected for a booster immunization. Spleen cells from the mice were fused with SP2/0 cells three days later. The positive hybridoma cells were screened by indirect ELISA with the recombinant p54 protein. The specificity of the selected mAb was further confirmed via Western blot and indirect immunofluorescence assay (IFA). The isotype of the mAb was detected using a subclass identification kit. Subsequently, the p54 protein was systematically truncated and expressed as GST fusion proteins to identify the antigen epitopes recognized by the mAbs by Western blot.

The constructed prokaryotic expression plasmid pET-21a-E183L (54-183 aa) was transformed into BL21 (DE3) cells. After induction with IPTG, the p54 recombinant protein was expressed in the supernatant in a soluble form with a molecular weight of about 17 kDa. Immunization of mice with purified p54 recombinant protein results in a serum titer of 1∶409 600 seven days after the third immunization. Cell fusion was successfully performed. After four rounds of subcloning, a hybridoma cell line named 5B11 that could stably secrete mAb against p54 protein was obtained. The ascites fluid was prepared and the mAbs were purified. Western blot and IFA assay results showed that 5B11 could specifically recognize p54 protein expressed in HEK293T cells and ASFV-infected porcine alveolar macrophages (PAMs). The mAb subclass identification showed that 5B11 was of IgG1 heavy chain and κ light chain. The epitope sequence recognized by 5B11 was ⁸⁰VTPQPGTSKPA⁹⁰.

In this study, the recombinant protein ASFV p54 with amino acid 54-183 was successfully expressed in a soluble form in the prokaryotic system. The development of anti-p54 mAbs and the identification of their recognized epitopes have expanded understanding of p54 protein epitopes and provided the basic materials for the serological detection of ASFV.