Construction of capsid VP2 mutants
Ten mutants with amino acids deleted from the N-terminal region of the VP2 capsid protein and three site-directed mutations in VP2 were constructed using the E. coli expression system (Fig. 1). The VP2 gene was truncated at the N-terminal by inserting PCR-amplified fragments flanked by Nde I and Xho I restriction enzyme sites into the pET28a vector (Novagen, Malaysia) described previously , as indicated in Table 1. Recombinant vectors were confirmed by using enzyme digestion and DNA sequencing (Sangon Biotech, Shanghai, China). Three site-directed VP2 mutants were obtained in vitro by using the Fast Site-Directed Mutagenesis Kit (Tiangen, Beijing, China). The pET28a plasmid, containing the full-length VP2 gene (1737 bp), was amplified by three pairs of mutation primers (Table 1). Briefly, amplification reactions were carried out in a total volume of 50 µL, containing 2 µL of template DNA, 2 µL of each forward and reverse primer (10 µM), 10 µL of 5× Fast Alteration Buffer, 1 µL of Fast Alteration DNA Polymerase (1.0 U/μL), and 33 µL of RNAse-free water. Polymerase chain reaction (PCR) procedure was followed as described below. Initial denaturation was at 2 minutes at 95°C followed by 18 cycles, each cycle consisting of 20 seconds at 94°C, 10 seconds at 55°C and 2.5 minute at 68°C. The final extension was 5 minutes at 72°C. After amplification, the PCR product was digested with Dpn I restriction enzyme at 37°C for 1 hour, and then 5 µL of the digestion product was transformed into FDM competent cells to screen for mutants. Desired mutants were identified by DNA sequencing.
Expression and purification of mutated VP2 protein
Full-length and mutant VP2 proteins were expressed in the E. coli system. Cells were grown in Luria-Bertani (LB) media supplemented with chloramphenicol 25 mg/L, kanamycin 50 mg/L, and L-Arabinose 2 g/L. Then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.15 mM to induce recombinant protein expression. After culturing the cells for 12 hours at 25°C at 200 rpm, the cells were harvested by centrifugation, resuspended in buffer A (150 mM NaCl, 50 mM Tris, pH 8.0), and lysed by ultrasonic treatment on ice. The expression of target recombinant protein was detected by western blotting (WB).
VP2 and mutant VP2 protein in the supernatant of E. coli cells were purified by a two-step method, using Ni-NTA affinity chromatography (Merck, Darmstadt, Germany) followed by SE-HPLC. Clarified cell lysates were pumped onto a Ni-NTA column balanced with 10 bed volumes of buffer A. After washing with 10 bed volumes of buffer B (150 mM NaCl, 50 mM Tris, 25 mM imidazole, pH 8.0), VP2 or mutant VP2 proteins were eluted with buffer C (150 mM NaCl, 50 mM Tris, 150 mM imidazole, pH 8.0). Fractions were further separated by SE-HPLC, using a Superdex™ 200 10/300 GL (GE Healthcare, Pittsburgh, PA, USA) column as described below. A Superdex™ 200 10/300 GL column was equilibrated with 10 bed volumes of buffer A at a rate of 1 mL/min. Subsequently, samples were applied to the Superdex™ 200 10/300 GL column. After binding, fractions were further eluted with buffer A. Elution fractions were collected and determined by 12% (v/v) SDS-PAGE. Fractions containing VP2 protein or mutant VP2 proteins were filtered with a 0.22 µm filter (Merck Millipore, Darmstadt, Germany) and quantified using a Micro BCA™ protein assay kit (Solarbio, Beijing, China). Importantly, all purification procedures were carried out at 4°C, and purified fractions were stored at −80°C to retain biological activity.
SDS-PAGE and western blotting analysis
SDS-PAGE and western blotting were conducted to detect protein expression as described below. First, 5× loading buffer was added to each cell lysate or fraction. Then mixtures were heated at 100°C for 10 minutes, resolved by 12% SDS-PAGE, and finally stained with Coomassie blue (Beyotime, Shanghai, China). For WB, samples were transferred from SDS-PAGE to PVDF membranes (Bio-Rad, Richmond, California, USA). VP2 or mutant VP2 proteins were detected using a horseradish peroxidase (HRP)-conjugated anti-his monoclonal antibody (Jackson ImmunoResearch, Philadelphia, PA, USA) at a dilution of 1:5,000. Membranes were observed with enhanced chemiluminescent reagent (NCM Biotech, Suzhou, China) or 3-amino-9-ethylcarbazole solution (ZSGB-BIO, Beijing, China), and examined by light microscopy (Leica DMI3000B) (Feica Microsystems, Wetzlar, Germany).
Supernatants containing VP2 or mutant VP2 were diluted with PBS for standard HA assays. To perform a HA test, 50 μL of clarified cell lysates containing VP2 or mutant VP2 were 2-fold diluted with PBS in a 96-V-shaped-well microtiter plate. Then 50 μL of guinea pig erythrocytes (0.8%) were added to each well. The plate was incubated at 37°C for 1 hour. HA titers were defined as the highest dilution that completely agglutinated guinea pig erythrocytes.
Indirect enzyme-linked immunosorbent assay
An indirect enzyme-linked immunosorbent assay was carried out using the conformational antibody 10A9, which only recognizes assembled capsids, as prepared in our previous study. First, 96-well plates were coated with purified mutant VP2 proteins, wild-type VP2 protein, or pET 28a empty vector, and then blocked with 5% skim milk in PBST at 4°C at least 8 hours. The conformational antibody 10A9 was used as the primary antibody and added into 96-well plates at 37°C for 30 minutes. After washing with PBST for five times, an HRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, Philadelphia, PA, USA) was used as the secondary antibody and incubated in the plate at 37°C for 45 minutes. Finally, antibody binding was analyzed by adding TMB solution (Solarbio, Beijing, China), and 2 M H2SO4 was added to stop the reaction. The optical density at 450 nm was recorded using a microplate reader (Omega Bio-Tek, Inc., Norcross, GA, Germany). All the experiments were performed in triplicate.
Transmission electron microscopy analysis
Transmission electron microscopy analysis was performed using a negative-staining method. Purified proteins were stained with 2% phosphotungstic acid (Solarbio, Beijing, China) for 1 minute and air-dried for 5 minutes at room temperature after removing excessive sample with filter paper. Grids were observed and imaged using a JEM-1400 (JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV, with a magnification of 120,000×.
Dynamic light scattering assay
A dynamic light scattering assay was measured at room temperature using the Nano Particle Analyzer (Malvern, Beijing, China). Samples were diluted to an identical concentration of 0.1 mg/mL before measurement, and all determinations were conducted at a scattering angle of 90° and equilibrated to 25°C. At least 20 acquisitions were collected for each sample. This assay was performed in triplicate for data analysis.
Sucrose gradient sedimentation analysis
VP2 and mutant VP2 proteins were purified in sucrose gradients. A four-step gradient of 5%, 15%, 30%, and 45% (wt/vol) sucrose was prepared in Tris-HCL buffer. All gradients were fractionated by bottom puncture, prepared in 12-mL tubes by loading 2 mL/step, and incubated at 4°C at least 8 hours to allow diffusion into a continuous gradient. Samples were firstly precipitated with 40% saturated ammonium sulfate (Sinopharm, Beijing, China), dialyzed with Tris-HCL buffer, loaded onto the gradients, and then ultracentrifuged at 200,000 ´ g in a P40ST rotor at 4°C for 36 hours (Hitachi, Ibaraki, Japan). After ultracentrifugation, samples in each fraction were electrophoresed via SDS-PAGE and identified by WB as described above. Native morphology of these samples was further examined using the NativePAGE™ Novex® Bis-Tris Gel System (Thermo Fisher Scientific, Waltham, MA, USA), and identified by LC-MSMS analysis (Sangon Biotech, Shanghai, China).