Identification of Linear B Cell Epitopes on CD2V Protein of African Swine Fever Virus by Monoclonal Antibodies

Figures

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  FIG 1. Expression and purification of the CD2V recombinant protein in the Bac-to-Bac baculovirus system. Results of SDS-PAGE (A), Western blotting (B), and IFA (C) indicated that CD2V recombinant protein was expressed well in the Bac-to-Bac baculovirus system. Results of SDS-PAGE (D) and Western blotting (E) for purification of the CD2V recombinant protein, respectively, showed that CD2V recombinant protein was purified by Ni-affinity chromatography. (F) Dot-ELISA for CD2V recombinant protein purification. (G) The molecular weight of CD2V protein was 46 kDa by size exclusion chromatography. (H) The CD2V protein was collected to 10 nm nanoparticles by 120 kV TEM. (I) Particle size of CD2V was approximately 10 nm in solution by Zeta CAD. (J) The reaction titer of the CD2V protein with the positive serum was 1:6,400 by indirect ELISA.

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  FIG 2. Titers and isotype identification of mAbs. (A) Titers of five mAbs, 18A3, 22B3, 43C2, 7E12, and 13G11, were 1:1.024 × 106, 1:1.28 × 105, 1:6.4 × 104, 1:1.28 × 105, and 1:6.4 × 104, respectively. (B) The heavy- and light-chain isotypes of 18A3 were IgG1 (OD450 of 1.016) and kappa (OD450 of 1.094). (C) The heavy- and light-chain isotypes of 22B3 were IgG1 (OD450 of 1.343) and kappa (OD450 of 1.179). (D) The heavy- and light-chain isotypes of 43C2 were IgG1 (OD450 of 1.206) and kappa (OD450 of 1.151). (E) The heavy- and light-chain isotypes of 7E12 were IgG1 (OD450 of 1.869) and kappa (OD450 of 1.209). (F) The heavy- and light-chain isotypes of 13G11 were IgG1 (OD450 of 1.489) and kappa (OD450 of 1.070).

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  FIG 3. Detection of the reaction between the mAbs and the CD2V recombinant protein using Western blotting and IFA. The 7E12 mAb could react with the CD2V recombinant protein by Western blotting (A) and IFA (B). The 22B3 mAb could react with the CD2V recombinant protein by Western blotting (C) and IFA (D). The 13G11 mAb could react with the CD2V recombinant protein by Western blotting (E) and IFA (F). The 18A3 mAb could react with the CD2V recombinant protein by Western blotting (G) and IFA (H). The 43C2 mAb could react with the CD2V recombinant protein by Western blotting (I) and IFA (J). Positive serum reacted with the CD2V recombinant protein by Western blotting (K) and IFA (L).

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  FIG 4. IPMA immunoperoxidase monolayer assay (IPMA) for CD2V mAbs. CD2V mAb 18A3 (A), 22B3 (B), 43C2 (C), 7E12 (D), and 13G11 (E) as well as ASFV-positive serum (F) could bind specifically to ASFV HLJ/18 strain. The negative control was pig negative serum, the blank control was PBS, and pig negative serum (G) and PBS could not react with ASFV HLJ/18.

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  FIG 5. Preliminary identification of CD2V protein B cell epitopes by the scanning peptide method. Reactions of CD2V mAbs with 18 short peptides were detected by i-ELISA (A to E) and dot-ELISA (G to K). (F) WH303 mAb was the negative control. Each experiment was conducted three times, and the error bars represent standard deviation (SD).

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  FIG 6. Truncation of short peptides. (A) Secondary structure analysis of peptide no. 14 by DNASTAR software. (B) Secondary structure analysis of peptide no. 18 by DNASTAR software. Reaction of the mAb 18A3 with two short peptides, no. 14 and no. 18, by i-ELISA (C) and dot-ELISA (E). Reaction of the mAb 7E12 with two short peptides, no. 14 and no. 18, by i-ELISA (D) and dot-ELISA (F). All the experiments were repeated three times and the error bars refer to standard deviation (SD).

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  FIG 7. Short peptides designed by overlapping polypeptide method to the extracellular region of CD2V. (A) Analysis of full-length sequence of CD2V protein. (B) Eighteen short peptides designed to the extracellular region of CD2V and sequences of no. 14 peptide and no. 18 peptide.