2. Materials and methods
2.1 Strains, reagents, cell
lines
The SP2/0 myeloma cells were kept
by the Henan Provincial Key Laboratory of
Animal Immunology (Zhengzhou, China), and SP2/0 cells were cultured in
Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco) medium
supplemented with 10% fetal bovine serum (FBS, Gibco, USA).
ASFV-positive sera (Convalescent sera from surviving pigs naturally
infected with ASFV) were a kind gift from Professor Hu of Institute of
Military Veterinary Medicine, Academy of Military Medical Sciences,
Changchun, China. Human embryonic kidney 293T (HEK293T) cells were
obtained from ATCC (Manassas, VA, USA) and stored in DMEM medium
(Solarbio, Beijing, China)
supplemented with 10% (v/v)fetal bovine serum (FBS, Gibco, USA).Escherichia coil BL21 (DE3) competent cells were purchased from
Takara Biomedical Technology (Beijing China). Animals care and study
procedure followed the guideline of the Animal Research Ethics Board of
Zhengzhou University.
2.2. Expression and purification of the ASFV p30 recombinant
protein
In response to efficient expression, we constructed three co-expression
system with chaperone plasmids. The chaperone plasmids include pKJE7,
pGro7 and pTf16. And then chosen the best one. Eventually we found that
0.1 mmol/L of IPTG, 12 h of expression time, and 18 ℃ of the
temperature, 0.4 mg/mL of L-arabinose are the best condition to obtain
the highest yield of soluble p30 protein.
The soluble recombinant protein was
purified by His Trap FF and His Trap Q HP affinity column.
The purified p30 protein was mixed with 5 × loading buffer, the mixture
was boiled for 10 min. Following separation by sodium dodecyl
sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), proteins were
transferred onto a polyvinylidene fluoride membrane. The membrane was
blocked overnight with 5% skim milk at 4 ℃ and incubated with anti-His
tag mAb (Solarbio, Beijing, China) 1:2000 for 1 h. After washing three
times with PBST, and then probed with a 1:2000 HRP-conjugated goat
anti-mouse IgG antibody at 37 ℃ for 1 h. The reactivity was visualized
using AEC reagents.
2.3. Construction of
pcDNA3.1-p30
According to the genome information of strain BA71V
(GenBankYP_009704045) Georgia 2007/1, the full-length ASFV p30 protein
gene with Bam HⅠ and Xho Ⅰ restriction sites was synthesized
and subcloned into the pcDNA3.1 vector (Invitrogen, Shanghai, China).
The recombinant construct was named pcDNA3.1-p30 and confirmed by PCR
and sequencing.
2.4. Monoclonal antibody generation and
characterization
Monoclonal antibodies against p30 were produced as described below.
Briefly, female, 6 weeks old, BALB/c mice were immunized subcutaneously
on week 0 with 50 μg purified p30 mixed with the same amount of complete
Freund’ adjuvant.
Two booster immunizations with p30
emulsified in incomplete Freund’ adjuvant were
administrated on weeks 2 and 4. The mice were administered 100 µg of p30
protein without adjuvant intraperitoneally. Mice were euthanized 3 days
later after the last administration, and harvested spleen cells were
fused with SP2/0 cells using polyethylene glycol (PEG 1500). After
HAT/HT medium selection, positive hybridomas cells were screened by
ELISA. Then, the supernatant was collected to identify p30-specific
antibodies with ELISA assay. Positive clones were sub-cultured three
times. The selected clones were cultured in the peritoneal cavities of
BALB/c mice primed with paraffin to obtain ascites fluid.
2.5. Indirect enzyme-linked immunosorbent
assay
Ninety-six-well plates were coated overnight at 4 ℃ with the purified
p30 protein (4 µg/mL, 100 µL/well) in 0.05 M carbonate-bicarbonate
buffer (CBS, pH 9.6). After washing with PBST (1×PBS with 0.05% Tween
20, pH 7.4), the plates were incubated with 5% skim milk at 37 ℃ for 2
h. Then, the first antibodies diluted with PBST were incubated at 37 ℃
for 30 min. After the incubation, 100 µL/well of HRP-conjugated goat
anti-mouse IgG antibody was added at a dilution of 1:5,000, and the
sample was incubated at 37 ℃ for
30 min, followed by washing three times. Tetramethylbenzidine (TMB) was
added and the signal was collected at 450 nm.
2.6. Immunofluorescence assay
(IFA)
HEK293T cells were seeded into 96-well cell culture plates at a density
of 4×104 cells/well and incubated overnight at 37 ℃
with 5% CO2. When the cells were 70-80% confluent, the
recombinant plasmid pcDNA3.1-p30 was transfected into the cells using
Lipofectamine® 2000 and cultured at 37 ℃ for 48 h. After cultured, the
plates were fixed with methanol containing 3% paraformaldehyde and
permeabilized with 0.3% Triton X-100 for 15 min at room temperature
(RT). Next, the plates were blocking with 5% skim milk at 37 ℃ for 2 h.
After three times washes, anti-p30 mAbs were incubated at 37 ℃ for 30
min, the plates were washed three times with PBS. Subsequently, the
plates incubated with FITC labeled goat anti-mouse IgG (1:100 in PBS)
for 30 min at 37 ℃. Cells were simultaneously stained by
4’,6-diamidino-2-phenylindole (DAPI, Solarbio,
Beijing, China). After the final wash, the fluorescence signals were
visualized by fluorescence microscopy. Mice immune and pre-immune sera
were used as positive and negative controls.
2.7. Peptide design and
synthesis
Bioinformatics tools were applied to analyze the entire sequence of p30
protein (201 aa). The secondary structure prediction was performed on
Ramachandran server
(http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html).
The B cell epitope regions were predicted using the online tool BepiPred
(BepiPred-2.0
(dtu.dk), and the epitope threshold was set up at 0.5 as previously
reported(Petrovan et al., 2019). The cleavage sites were selected in the
rigid regions to conduct overlapping peptides of p30 protein without
destroying the protein flexible structure. Based on a comprehensive
consideration of bioinformatics prediction, 13 overlapping peptides
(with an offset of 5 amino acids) covering the entire p30 protein and
the further truncated peptides were synthesized by GL Biochem (Shanghai,
China).
2.8. Dot-blot assay and peptide
ELISA
Dot-blot hybridization for identification of linear epitopes was based
on the method described by previously described method(Chen, Wu, Huang,
Cheng, & Chang, 2015). Dot immunoblotting assay was employed to
determine the epitopes recognized by mAbs. Polypeptide were prepared
dissolving 1mg of solid in 100 µL of DMF (HPLC grade), 2.5 mg of bovine
serum albumin (BSA) was mixed with 1.5mg of EDC in 1.25 mL of PBS. And
then, the above solution was mixed slowly and after 10 min, 0.5mg of EDC
was added, and stired at 4 ℃ for 18 h. The reacted mixture was dialyzed
for 3 days. Centrifuged at 3500 r/min for 10 min, the supernatant was
kept at -20 ℃. ASFV p30 protein peptides were spotted onto
nitrocellulose membranes (Pierce, USA), and blocked with 5% skim milk
in PBST at 4 ℃ overnight. After washing 5 times with PBST, the membranes
were incubated with anti-p30 antibody at 37 ℃ for 1 h. HRP-conjugated
goat anti-mice IgG antibody was used as the secondary antibody, and the
results were observed by incubating with AEC reagent.
In peptide ELISA, 96-well plates were coated with 6 µg/mL peptide-BSA
conjugates (100 µL/well) in 0.05 M CBS buffer (pH 9.6) at 37 ℃ 2 h.
Then, the plates were blocked with 5% skim milk in PBST. Next, mAbs
anti-p30 were used as the primary antibodies. BSA was used as negative
control. HRP-conjugated goat anti-mouse IgG was used as the secondary
antibody. The reactions were developed using TMB. The OD values of each
well were measured at 450 nm using an ELISA microplate reader.
2.9. Reactivity of the linear B cell epitopes with
ASFV-positive
serum
To assess whether the linear B cell epitopes could be recognized by
ASFV-positive serum, the BSA and peptides were then conjugated using the
EDC reaction. The reactivity of the synthesized peptides with
ASFV-positive swine serum was determined by the above-mentioned
dot-blot.