3. Results
3.1. Expression and purification of the recombinant p30
protein
Target proteins were purified from the supernatants of bacterial lysis
product and analyzed by means of SDS-PAGE and Western blot assay. As
shown in Fig. 1a and Fig. 1b, p30 protein was successfully expressed,
with molecular weights of 34 kDa. The Western blot (Fig. 1b),
demonstrates that the purified p30 protein could be recognized by
anti-His tag mAb.
3.2. Screening and features of mAbs against p30 protein
To generate anti-p30 mAbs, two female BALB/c mice were separately
immunized with purified p30 protein at two-week interval. The two mice
with the higher antibody titers were used to generate mAbs by the
hybridoma technique. ELISA and western blotting results showed that all
the mAbs specifically recognized p30 protein (Fig. 2a - b). Supernatants
from the resulting hybridoma cells were screening yielded eleven primary
hybridoma clones, which were subcloned to obtain 3 monoclonal cell
lines, and named as 7D2, 2F6, and 8C8. Results from IFA further
confirmed the reactivity binding of these mAbs to p30 protein (Fig. 3).
3.3. Mapping of the linear B-cell epitopes of p30 protein
using
mAbs
To identify specific B-cell epitope, 13 overlapping polypeptides from
ASFV p30 protein were designed with an offset of 5 amino acids (Fig.
4a). We designed p30 fragments from the CP204L based on in silico
predicted B-cell linear epitopes obtained using BepiPred.
Results from dot-blot and peptide
ELISA indicated that 2F6 and 8C8 reacted only with the L1 (1-20 aa), and
7E2 reacted only with L2 (16-35 aa). And then 3 overlapping polypeptides
from L2 were designed with an offset of 5 amino acids (Fig. 4b). Three
overlapping polypeptides from L1 were designed with an offset of 5 amino
acids (Fig. 4c). Results from dot-blot and peptide ELISA indicated that
residues 26-35 aa (L2-3) (Fig. 4c).
The identified epitope regions were further truncated as shown in the
Fig. 4b. The details of the truncated peptides are listed in S1.
Dot-blot and peptide ELISA results suggested that the linear B cell
epitopes on ASFV p30 protein were located at
residues1MDFILNISMKMEVIFKTDLR20and 26 VFHAGSLYNW35.
3.4. Biological information analysis of the identified
epitopes
To deeply analyze the conservation of the identified epitopes among
different strains, we analyzed p30
sequences from 19 genotypes(Petrovan et al., 2019) (Table S2). In the
epitope 1MDFILNISMKMEVIFKTDLR20, an
amino acid substitution (M→V) at site 9 was observed only in one
(GenBank accession ACJ61516.2) of 19 sequences. In the epitope26VFHAGSLYNW35, the amino acid
alignment identified a total of 5 aa differences in the mAb 7D2 epitope
region. One single aa difference, His28 to
Asn28 between
Georgia/07 and Genotype XIII, an
amino acid substitution (N→T) at
site 34 was observed in four genotype (GenBank accession ACJ61560.1,
ADQ44025.1, ACJ61548.1, ACJ61546.1 ). The result implied that the26VFHAGSLYNW35 might be less
conserved than the epitope1MDFILNISMKMEVIFKTDLR20.
Furthermore, p30 proteins of 19 representative p30 strains (Table S2).
These results further confirmed that the two linear B cell epitopes
identified in our work were highly conserved in diverse p30 strains
(Fig. 5a-b).
To understand the structural mechanism of the epitopes identified by
these three mAbs, the 3D structure was predicted using an online
computer software program Phyre2
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index)(Kelley,
Mezulis, Yates, Wass, & Sternberg, 2015). The result is shown in the
Fig. 5c. Furthermore, the 3D model of p30 was obtained after molecular
dynamics simulation and energy minimization. The visualization software
PyMol was used to view the position of predicted linear epitopes and
digestion-resistant peptides of p30 in the tertiary structure. To
understand the spatial distribution of the identified epitopes, the 3D
models of p30 protein used for further analysis. As shown in Fig. 6c,
all the identified linear epitope26VFHAGSLYNW35and 1MDFILNISMKMEVIFKTDLR20 were
exposed on the surface of the p30 protein (Fig. 5d).
3.5. Reactivity of the linear B cell epitopes with ASFV
positive
serum
Recognition of the epitope peptides by
ASFV-positive serum were tested by
dot-blot and ELISA. Results showed that the epitope26VFHAGSLYNW35and “1MDFIL
NISMK MEVIF KTDLR20” could be recognized by
ASFV-positive serum. These results suggested that epitope26VFHAGSLYNW35 and1MDFIL NISMK MEVIF KTDLR20 may be
immunodominant epitopes (Fig. 6a-b).