Novel Epitopes Mapping of African Swine Fever Virus CP312R Protein Using Monoclonal Antibodies

Figures

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  Figure 1. The recombinant CP312R expression in prokaryotic cells and its immunogenicity analysis using sera from pigs infected with the ASFV/HLJ/HRB1/2020 strain. Analysis of the recombinant CP312R protein expression by Western blotting assay using an anti-His-tag antibody (A). The AKAT affinity-based liquid chromatography purification by using a 6x-His Trap HP column process revealed a single peak in the 85–110 mL elution volume. The purified recombinant CP312R protein was subjected to SDS-PAGE for CBB (B). The target protein’s immunogenicity was evaluated using the iELISA and Western blot tests (C), with ASFV-positive pig sera (n = 36, pigs infected with the Pig/HLJ/HRB1/2020 strain) and SPF pig controls (n = 12, pigs free from the ASFV).

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  Figure 2. Monoclonal antibody production and isotyping. iELISA and Western blotting analyses were used to screen the mouse for an immune response against the target protein (A). A single hybridoma clone’s reactivity analysis was performed by iELISA on the recombinant CP312R protein (B). The generated mAbs were isotyped into their immunoglobulin (IG) class (heavy or light chains) by using the Pierce Rapid ELISA Mouse mAb Isotyping Kit (ThermoFisher Scientific) (C). M-1 = Mouse number one, M-2 = Mouse number two, M-3 = Mouse number three.

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  Figure 3. The specificity analyses for mAbs. The IFA analyses were performed in HEK-293T cells transfected with the plasmid of CP312R (1 µg/well, 12-well plate) (A) and in PAMs infected with Pig/HLJ/2018 isolate (MOI = 1) (B) to observe the CP312R-mAbs’ specific interaction. The generated mAbs interacted with the CP312R protein expressed in various cells infected with the ASFV and lentivirus and transfected with the plasmid CP312R (C). The anti-ASFV CP312R mAbs’ specificity was evaluated using a Western blotting assay (D) and an iELISA (E). The 96-well plates were coated with the purified A104R, PLCP, porcine-HEV, and swine MBP proteins (0.2 µg/mL). Lane 1 = A104R protein, lane 2 = PLCP (Papain-Like Cysteine Protein), lane 3 = Porcine-HEV (Porcine Hepatitis E virus), lane 4 = Swine MBP (Myelin Basic Protein), lane 5 = CP312R protein, WSL=Fetal wild boar lung cell line.

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  Figure 4. The CP312R antigenic determinants are identified by using the characterized mAbs. The schematic representation of the ASFV CP312R and its truncated fragments in the epitope identification process. The numbers in the boxes represent the positions of the amino acids (AAs) in the CP312R protein as well as the truncated fragment protein (A). Identification of the recognizable truncated region using the seven characterized mAbs by Western blot (B). Further identification of the epitopes in the recognized truncates using the characterized mAbs by Western blot (C). IFA analysis of the identified epitopes by the defined mAbs (D).

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  Figure 5. Conservation analysis of newly identified epitopes of the CP312R protein. Sequences of the CP312R proteins from 21 various ASFV strains belonging to eight genotypes were obtained from NCBI database, and aligned using DNAStar software. The red boxes represent the identified epitope sequences. The differently colored amino acids are presented as unconserved amino acids in comparison to the main protein core.

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  Figure 6. ASFV CP312R subcellular localization analysis in WSL and PAMs infected with the lentivirus-CP312R and ASFV HLJ/HRB1/2020, respectively.