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.