Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

Abstract

African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (³⁶⁶ANRERYNY³⁷³), epitope 2 (⁴¹⁵GPDAPGLSI⁴²³), and epitope 3 (⁴⁹⁸EMLNVPDD⁵⁰⁵). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

Fig. 1

Schematic diagram of epitopes recognized by the mAbs. (A) Full-length pB602L protein of ASFV strain Pig/HLJ/2018. (B) Design of polypeptide fragments (S1-S18) in pB602L protein and the position of the three epitopes (³⁶⁶ANRERYNY³⁷³, ⁴¹⁵GPDAPGLSI⁴²³, ⁴⁹⁸EMLNVPDD⁵⁰⁵). (C) List of mAbs reacting with the corresponding pB602L regions

Fig. 2

Expression and purification of pB602L. (A) SDS-PAGE analysis of pB602L expressed in E. coli. (B) Identification of pB602L using an anti-GST antibody. (C) Identification of pB602L using an anti-ASFV serum. M, protein molecular weight marker; lane 1, cells transfected the empty plasmid pGEX-6P-1; lane 2, uninduced cells transfected the recombinant plasmid pGEX-6P-B602L; lane 3, total cell lysate prepared from cells induced with 0.3 mM IPTG; lane 4, purified pB602L without GST tag

Fig. 3

Detection of the pB602L protein using IFA. Primary PAMs infected with ASFV (Pig/HLJ/2018) at an MOI of 0.2 were harvested 24 h postinfection and fixed in 4% paraformaldehyde at 4°C for 24 h, followed by permeabilization with 0.1% (w/v) Triton X-100 at room temperature for 10 min. Representative mAbs of the three groups – B2E12, B2H10, and B2E12 – were incubated with the PAMs infected with ASFV. As a positive control, PAMs were incubated with an anti-porcine ASFV serum. The scale bar represents 50 μm

Fig. 4

Immunohistochemical assay for detecting pB602L in ASFV-infected pig tissues. Tissues, including the spleens, tonsils, and gastrohepatic lymph nodes, were collected from pigs that were infected with 10^2.5 times the 50% hemadsorbing dose (HAD₅₀) of ASFV strain Pig/HLJ/2018 through intramuscular injection and euthanized on day 7 postinfection. These tissues were fixed, embedded, and sliced using a standard protocol. Three mAbs (B2A1, B2H10, and B2E12) were used as the primary antibodies to stain the pB602L protein in ASFV-infected cells. The scale bar represents 50 μm