Identification of a novel linear B-cell epitope on the p30 protein of African swine fever virus using monoclonal antibodies

Fig. 1. Expressed and purified ASFV p30 protein and recombinant p30 immunogenicity analyzes. A: Expressed of p30 protein. Lane M: protein marker (10∼180 kDa); Lane 1: uninduced E. coli BL21 culture; Lane 2: induced E. coli BL21 lysate; Lane 3: soluble fraction. B: WB was used to analyze recombinant HIS-tag p30 protein reactivity. Lane M: protein marker (10∼180 kDa); Lane 1: Non-induced E.coli BL21 Lysate; Lane 2: purified p30 protein. C: Purified of p30 protein. Lane M: protein marker (10∼180 kDa); Lane 1: the purified p30 protein. D: Target proteins was examined using WB. Lane M: protein marker (10∼180 kDa); Lane 1: empty pET30a plasmid; Lane 2: purified p30 protein.

Fig. 2. Production of McAb against p30. (A) Potency of mouse serum. (B) Isotype determination of McAb lB4G2–4. (C) The potency of the McAb 1B4G2–4.

Fig. 3. McAb lB4G2–4 specificity analyzes using WB and IFA. (A) Western blotting results for McAb against p30 protein. Lane 1: the unrelated protein pB602L with His-label served as an invisible control protein with his tag; Lane 2: p30 protein. (B) Results of IFA testing of McAb 1B4G2–4 against p30 protein. (C) McAb lB4G2–4 specifically bound to the ASFV HLJ/2018 strain. Lane M: protein marker; Lane 1: PAM cells not infected with ASFV; Lane 2: Inactivated samples of ASFV Pig/HLJ/2018 strain-infected PAM cells.

Fig. 4. Blocking/competitive ELISA detection of positive anti-ASFV sera against p30 McAb; (A) negative anti-ASFV sera were used to determine baseline OD450 values in ELISA; (B) data are presented as means ± standard deviations. Statistical significance was determined by paired t-tests. p < 0.01; ***.

Fig. 5. Design of truncated overlapping short peptides and preliminary identification of epitopes. The original entire p30 sequence is blue. (A) Schematic design of the truncated overlapping short peptide across the p30 protein. (B) and (C) Western blotting assay using the McAb 1B4G2–4 to identify the first recombinant peptide fusion protein.

Fig. 6. Identification of linear epitopes recognized by monoclonal antibodies 1B4G2–4. (A) Nine synthetic overlapping peptides spanning the P30 protein; (B) and (C) Dot blotting analyzes of McAb against different peptides. PBS was used as negative controls.

Fig. 7. Sequence conservation analysis and spatial structure distribution of the identified linear B-cell epitope. (A) Multiple sequence alignment of linear epitope 164HNFIQTI170 with p30 from other ASFV strains.164HNFIQTI170 is marked with a purple box. (B) Predicted structural model of ASFV p30. The structure of p30 protein is predicted by Phyre2 with a cyan-colored surface. 164HNFIQTI170 (purple) is shown on the p30 protein.