Cloning and Expression of a Truncated Form of the p72 Protein of the African Swine Fever Virus (ASFV) for Application in an Efficient Indirect ELISA System

Figure 1. (A) Amino acid sequence of African swine fever p72 protein (B646L) from the Georgia 2007 sequence (646 aa residues). (B) Amino acid sequence of a fragment selected as the best candidate for the expression of ASF-p72-truncated protein (73-254 aa residues). Hydrophobicity, antigenicity index, and surface probability are represented by the Kyte and Doolittle hydrophobicity plot (blue: hydrophobic regions), Jameson–Wolf antigenic index (pink), and Emini surface accessibility prediction (yellow); red boxes indicate protein sequence regions where the antigenic determinants were found using the Jameson and Wolf analysis (n = 10). (C) Prediction of two glycosylated sites in the protein fragment at 190 aa residue (blue lines). (D) Prediction of antigenic determinant in the ASF-p72-truncated protein (the same antigenic regions are marked in (B) with different colors).

Figure 2. (A) A proposed model of structural analysis. (B) Nine epitopes localization of ASF-p72-truncated protein.

Figure 3. Phylogenetic trees were created using the MEGA X software, with at least 1000 replicates, using the maximum likelihood based on the JTT model and gamma distribution. ▲ Strains reported in America; ● Reference strain Georgia 2007; ◆ Strains reported in China.

Figure 4. (A) PCR amplification of a fragment from the B646L gene by temperature gradient PCR. M: base pair marker 100 bp; lane 1: 63.5 °C; lane 2: 64.3 °C; lane 3: 67.6 °C; lane 4: 68 °C; lane 5: 71.7 °C. (B) pET-SUMO-ASF-p72-truncated protein plasmid. For the PCR reaction conditions, 68 °C is used. M: base pair marker 1 kb; lane 1/2: recombinant plasmid.

Figure 5. ASF-p72-truncated protein was expressed as inclusion bodies. Coomassie stain (A) and Western blot (B) notably show the protein in greater quantity, making it easy to identify. The considerable signal observed is indicative of the efficiency of the expression of recombinant protein at ~42 kDa expected molecular weight (arrow on lanes 3, 4, 5, and 6). M: marker; lane 1: negative control; lane 2: positive control (CAT protein/37 kDa).

Figure 6. Purified ASF-p72-truncated recombinant protein (ASF-p72t) from inclusion bodies by IMAC. Coomassie stain (A) and Western blot (B). (M: marker; lane 1: 5 µL of ASF-p72t; lane 2: 10 µL of ASF-p72t; lane 3: 15 µL of ASF-p72t).

Figure 7. Graph shows humoral immune response in mice immunized with ASF-p72-truncated recombinant protein (ASF-p72t) by iELISA. * p < 0.05.

Figure 8. High antibody detection capacity of the ASF-p72-truncated iELISA of serum dilution samples from the IRTA-CReSA OIE Reference Laboratory sera panel (Table 2).

Figure 9. Indirect immunoperoxidase test (IPT) confirmed the true negative (A) and positive (B) pig serum samples.

Figure 10. Serological distribution of positive and negative serum samples (A) tested by iELISA of ASF-p72-truncated protein. The dotted line represents the cut-off value for iELISA and was calculated as 0.239, testing 43/46 negative serum samples and 22/23 positive serum samples (B). Sera above the dotted line were considered ASF positive, and sera below the dotted line were considered ASF negative according to the cut-off value. Intra-assay (% CV) determination (C) using 43 negative sera.