Fig. 1 Schematic map of the reactions between the polypeptides and the mAbs. (A) Full-length pB602L protein of ASFV Pig/HLJ/2018; (B) Ploypetide fragments (S1-S18) from different regions of pB602L expressed in E.coli and the position of three epitopes; (C) mAbs of the three epitopes.
Fig. 2 Expression and purification of pB602L. (A) SDS-PAGE analysis of pB602L protein expressed in E. coli . M: Protein molecular weight marker; Lane 1: Empty pGEX-6p-1 transfected cells; Lane 2: Non-induced cells; Lane 3: IPTG induced cells; Lane 4: Purified pB602L with GST tag removed. (B) pB602L identified by an anti-GST antibody. M: Protein molecular weight marker; Lane 1: Empty pGEX-6p-1 transfected cells; Lane 2: Non-induced cells; Lane 3: IPTG induced cells. (C) pB602L protein identified by an anti-ASFV serum. M: Protein molecular weight marker; Lane 1: Empty pGEX-6p-1 transfected cells; Lane 2: Non-induced cells; Lane 3: IPTG induced cells; Lane 4: Purified pB602L with GST tag removed.
Fig. 3 Immunofluorescent assay detecting pB602L in ASFV-Pig/HLJ/2018 infected PAM cells. Primary PAMs were plated on 24-well plates and infected with ASFV at an MOI of 0.2. At 24 h postinfection, cells were fixed in 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.1% (w/v) Triton-100 for 10 min at room temperature. B2E12, B2H10 and B2E12 are mAbs as primary antibodies. Cells stained with mouse anti-ASFV serum as positive controls.
Fig. 4. Localization of pB602L protein with monoclonal antibodies against different epitopes in PAM cells infected with ASFV (Pig/HLJ/2018). Primary PAMs were plated on 6-well plates and infected with ASFV at an MOI of 0.2. At 24 h postinfection, cells were fixed in 4% paraformaldehyde for 10 min at room temperature . pB602L protein was detected by mAbs B2A1(A), B2H10(B), or B2E12(C) and visualized with a gold-labeled goat-anti-mouse IgG in an electron microscopy. The control (D) was stained with PBS instead of a mAb. White arrows point to gold particles indicating the locations of pB602L in a ASFV assembly factory. Scale bars represent 100 nm.
Fig. 5. Immunohistochemical detection of ASFV pB602L protein in tissues of pigs. B2A1, B2H10, and B2E12 are monoclonal antibodies against different epitopes of pB602L protein. The spleens, tonsils, and gastrohepatic lymph nodes were collected from mock or ASFV (Pig/HLJ/2018) infected SPF pigs.