2. Materials and methods
2.1 Design and synthesis of primers and
genes
The gene sequence of BTV-1 VP3 (GenBank accession No. MG255581.1) was
codon-optimized for E.coli preference codons. The VP3 sequence
primers of EcoR I and Xho I were designed in Primer 5.0:
VP3-F: CGGAATTCATGGCCGCTCAGAACGAAC; VP3-R: CCGCTCGAGTTACACTGTTGGGGCAGCC,
and synthesized by Shanghai Shenggong Biological Co., Ltd. The BTV-1 VP3
plasmid was used as a template for PCR and the recovery of the target
gene.
The fusion protein NusA sequence conteined in the vector pET-43.1a was
used as a template and the fusion protein primers of Nde I andBamH I were designed in Primer 5.0 (the thrombin sequence was
inserted after the downstream primer cleavage site): NusA-F:
GGAATTCCATATGATGAACAAAGAAATTTTGGCTGT; NusA-R:
CGGGATCCCTGGTGCCACGCGGTTCTCGCTTCGTCACCGAACCAG.
2.2 Culture of bacteria and construction of expression
vector
The pET-28a was selected and amplified in LB medium with kanamycin (50
μg/ml), and pET-43.1a, pTf16, and pET-32a were selected and amplified in
LB medium with ampicillin (50 μg/mL). The strains were preserved in the
Laboratory of Molecular Immunology at Zhengzhou University.
The NusA fusion protein was ligated to the pET-28a vector to construct a
new vector pET-28a-NusA. The VP3 gene was ligated to the pET-28a-NusA,
pET-28a, and pET-32a vectors, respectively. Thus far, we constructed 3
recombinant vectors: pET-28a-VP3, pET-28a-NusA-VP3 and pET-32a-VP3.
2.3 Protein expression identification and condition
optimization
Competent cells of E. coli BL21 (DE3) were prepared according to
the manufacturer’s protocol (TaKaRa, China). Subsequently, 3 plasmids
was transformed into the BL21(DE3) competent cells and the plasmid
pET-28a-VP3 was transformed into pTf16-BL21. Then, 4 kinds of expressing
bacteria were activated and expanded respectively. The
pET-28a-VP3-pTf16-BL21 was added with 0.5 mg/ml L-arabinose during
expansion culture. When the OD600 reached 0.6, 0.1 mM of
isopropyl β-D-thiogalactoside (IPTG) was added to induce expression for
12 h at 25 ℃. After the cells were harvested under centrifugation at
12000 rpm for 5 min at 4 ℃, every 0.01 g pelleted bacteria were
suspended in 150 μl Tris-HCl (25 mM Tris and 150 mM NaCl, adjust pH to
8.5 with HCl). Afterwards, the pelleted bacteria were lysed by
sonication in an ice water bath. 200 μl of the crushed liquid was
centrifuged at 12000 rpm for 15 min at 4 ℃. The supernatant and the
precipitate (resuspend the precipitate with 200 μl Tris-HCl) were
separated and identified by SDS-PAGE and Western-blot.
The expression conditions of the supernatant with highest expression
among four bacteria solutions were optimized with temperature ( 20 ℃,25
℃,30 ℃ and 35 ℃ ) , IPTG concentration ( 0.1 mM,0.3 mM,0.5 mM and 0.7 mM
) , L-arabinose ( 0.5 mg/ml,1.5 mg/ml,2.5 mg/ml and 3.5 mg/ml ) and time
( 6 h,9 h,12 h and 15 h ).
2.4 Purification of protein
The supernatant was collected after centrifugation at 12000 rpm for 15
min at 4 ℃ and filtered with a 0.45 μm filter. Then purified by Ni-NTA
affinity chromatography. More specifically, the supernatant was combined
with the Ni-NTA at 4 ℃ for 2 h, and then the binding buffer ((Tris-HCl,
pH 8.5) equilibrated Ni-NTA column. After washing with 10 beds of
washing buffer (Tris-HCl with 40 mM imidazole, pH 8.5), the recombinant
VP3 was eluted with elution buffer (Tris-HCl with 80 mM imidazole, pH
8.5). The collected protein was dialyzed with Tris-HCl for 24 h (the
Tris-HCl was changed every 3-4 h). The dialyzed protein was concentrated
with silica beads for 2-4 h, when it was 1/2 or 1/3 of the original one
in volume. Then we identified it by SDS-PAGE and used HRP Conjugated
Anti-His Tag Mouse Monoclonal Antibodies (Ybbkine®Biotechnology Co., Ltd.) for Western-blot tests.
2.5 Mice immunization
Two 6-8 weeks old BALB/c mice were selected and each mouse was immunized
with 30 μg of VP3. The protein and adjuvant are mixed at the ratio of
1:1 and emulsified before immunization. Freund’s complete adjuvant was
used for the first immunization on day 1, and Freund’s incomplete
adjuvant was used for the second immunization and third immunization on
day 21 and 42, respectively. The blood was collected from tail vein on
days 0 and 56 and stored at -20 ℃. Moreover, we used 50 μg of
inactivated BTV-1 virus mixed with adjuvant at the ratio of 1:1 and
emulsified before immunization. One week after the third immunization,
mouse blood was collected from tail vein and stored at -20 ℃. The
collected blood was used as positive control in Dot-ELISA test.
2.6 Indirect ELISA
The purified VP3 was mixed and diluted with carbonate buffer (pH 9.6),
and then coated in a 96-well plate at 100 μl/well (placed at 4 ℃ for 1
hour). The buffer was discarded and washed 3 times with PBST. After
that, 5% skim milk was added to the plate, 100 μl per well, and blocked
at 37 ℃ for 2 hours. Then, the blocking solution in the plate was
discard and the plate was washed 3 times with PBST. 200 μl of mouse
serum was diluted with blocking solution to 1/1000 and added to the
first well of the plate while 100 μl of blocking solution was added to
the rest wells. After mixing, 100 μl of mixture in the first well was
added to the second well. After mixing again, 100 μl of mixture in the
second well was added to the third well and incubated at 37 ℃ for 1
hour. The primary antibody was discarded and washed 3 times with PBST.
100 μl of HRP- conjugated goat anti-mouse IgG was added to each well and
incubated at 37 ℃ for 1 hour. The secondary antibody was discarded and
wash 5 times with PBST. 100 μl of coloring solution was added to each
well, and avoid light reaction for 5-10 min. Then, 100 μl of stop
solution (2 mol/L H2SO4) was added to
each well. When OD450 in well to be tested was 2.1 times
great than OD450 in NC well, the corresponding antibody
dilution factor is the antibody titer.
2.7 Dot-ELISA
First, a round hole of proper size was made on the NC membrane, and then
it is soaked with double distilled water and dried. Added 1-2 μl sample
into the circular hole. After being dried, the membrane was sealed with
5% skimmed milk (37℃ for 2 hours or 4℃ overnight). The blocking
solution was discarded and washed 3 times with PBST, and the primary
antibody (mouse anti-BTV-1 polyclonal sera) was incubated at 37℃ for 1
hour. After discarding the first antibodies, the plate was washed with
PBST for 3 times, the second antibody (HRP-conjugated goat anti-mouse
IgG) was incubated at 37℃ for 1 hour, and then washed with PBST for 3
times for AEC color reaction.
2.8 Indirect immunofluorescence assay
(IFA)
The optimized VP3 gene was digested with EcoR I and Xho Iand then cloned into pcDNA 3.1 to obtain the eukaryotic expression
recombinant plasmid pcDNA 3.1-VP3. After the 293T cell line was
recovered and subcultured with DMEM complete medium (10% serum and 90%
DMEM), the cells were seeded at a density of 1.5×104cells/well in the 96-well plate. When the cells were 70% confluent, the
plasmids pcDNA3.1-VP3 and pcDNA3.1 (BC) transient transfect into cells
by transfection kit (jetPRIME® in vitro DNA & siRNA
transfection reagent PROTOCOL). The cells were fixed with methanol at
room temperature for 15 min, and washed third with PBS. Mouse anti-VP3
polyclonal sera was added to each well at 1:200 dilution, and incubated
at 37℃ for 1 hour, and the plates were washed three times with PBS.
Then, fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG
(Sigma-Aldrich) (1:1000 dilution) was added, before incubation at 37℃
for 1 hour, and five times washes with PBS. The cells were then observed
under a fluorescent microscope.