Strains and plasmids
Laboratory strain K. marxianus Fim-1DURA3 is a uracil auxotroph strain originated from K. marxianus Fim-1 deposited in China General Microbiological Culture Collection Center (CGMCC No.10621 . The vector pUKDN115 was constructed from the pUKD-S-PIT plasmid by replacing the fragment containing an α factor signal peptide sequence and human interferon α-2a gene with a multiple cloning site (MCS) .
Construction of the recombinant strain
According to the K. marxianus codon preference, the VP2 gene of Kresse strain (GenBank U44978.1) was optimized and synthesized by Genewiz Biotechnology Co., Ltd (Suzhou, China). The optimized VP2 gene was deposited in the NCBI GenBank database under an accession number MT932328. The synthetic VP2 gene was amplified with Phanta® Super Fidelity DNA Polymerase (Vazyme, Nanjing, China) using the following oligonucleotide primers (underlining indicates the homologous sequences from pUKD-N115 vector): 5'-TTTTTTTGTT AGATCCGCGG ATGAGCGAAA ACGTGGAGC-3') and 5'-AGCTTGCGGC CTTAACTAGT CTAGTACAAC TTTCTTGGG -3'. The amplicon was ligated with the EcoR I and Hind III linearized pUKDN115 by Gibson assembly , and then directly transformed into the FIM-1 DURA3 strain according to the Lithium acetate transformation method . Transformants formed on the SD plates (0.67% YNB, 2% glucose, 2% agar) were verified by PCR using the primers ATGAGCGAAA ACGTGGAGC-3 and CTAGTACAAC TTTCTTGGG -3', and the positive clone was designated to the KM-PPV-VP2 strain.
Expression and identification of the VP2 Protein in K. marxianus
Fresh clones of KM-PPV-VP2 were inoculated in 50 mL YD medium (2% yeast extract, 4% glucose) and cultured at 30°C, 220 rpm for 72 h. One milliliter of yeast cells harvested by centrifugation was washed with 1 ml PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) twice, and then suspended in 500 μl lysate buffer (50 mM HEPS, 140 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % Na-Deoxycholate, pH 7.5). Approximately 400 μl of glass beads (G8772, Sigma-Aldrich, Missouri, USA) was added to disrupt cells on a Bead-beater (FastPrep-24, MP, California, USA) at 6 m/s for 2 min. Cell lysates were centrifuged at 12000 rpm, 4 °C for 20 min, and supernatants were used for SDS-PAGE and Western blotting assays. An anti-PPV VP2 polyclonal antibody and a goat anti-mouse IgG alkaline phosphatase-conjugate (074-1806, KPL, USA) was used as the primary and secondary antibody in western blotting respectively.
Preparation of the anti-PPV VP2 polyclonal antibody
The native VP2 gene of Kresse strain was cloned into a pET-28a (+) vector within the Sac I and Not I sites ( (Novagen, Madison, USA), generating the pET-28a/VP2 plasmid. After transformation into E. coli BL21(DE3), VP2 protein was induced by 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and purified by Ni-NTA (Ni Smart Beads, Smart-lifesciences, Changzhou, China) affinity basically as previously described . Six weeks old Balb/C mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, were immunized with 20 μg of purified VP2 antigen mixed with an equal volume of Freund's adjuvant. After 35 days post-immunization (dpi), sera were separated and used as a primary antibody for Western blotting described above.
Transmission Electronic Microscopy (TEM)
TEM scans of PPV VLPs were performed on a JEM-2100 Electron Microscope (JEOL Tokyo, Japan) according to Bucarey et a l . Briefly, samples were spotted onto carbon-coated copper grids. After adsorption at room temperature for 5 min, copper grids were dried with filters and negatively stained with 3% of phosphotungstic acid (PTA). The grids were examined at an accelerating voltage of 120 kV.
High cell-density fermentation
High cell-density fermentation was conducted in a 5 L fermentor (BXBIO, Shanghai, China) as described recently . The KM-PPV-VP2 strain was inoculated in 200 mL YD medium, grown at 30 °C, 220 rpm for 18 h, and then transferred into a fermentor containing 2 L defined mineral medium . During fermentation, the dissolved oxygen was maintained above 10%, and the temperature was controlled at 30 °C. The pH was controlled automatically at 5.5 with ammonium hydroxide. At given intervals, 10 ml of culture was harvested to determine the cell density (OD600 nm) and wet cell weight (WCW). For SDS-PAGE analysis of VP2 productions, cell samples were diluted 1:10 with PBS buffer before disruption using the glass bead disruption described above. VLPs quantification was performed on an Agilent Series 1100 System (Agilent, Waldbronn, Germany) using a TSKgel G4000 SWXL column (300mm x 7.8 mm i.d.) (Tosoh Bioscience, Stuttgart, Germany) and a TSKgel SWXL guard column (40.0 mm x 6.0 mm i.d.) (Tosoh Bioscience) as previously described .
Screen of the ion-exchange chromatography (IEX) media
KM-PPV-VP2 cells were collected by centrifugation at 5, 000 rpm for 10 min, followed by washing with deionized water twice. Rinsed cells were then suspended with PBS buffer pH7.4 for cation exchange, or with 20 mM Tris-HCl buffer pH 8.0 for anion resins. Cell lysates were prepared by high-pressure homogenization on a JN-02C Homogenizer (JNBIO, Guangzhou, China) under a condition of 1500 bar, 4 °C for 2 times, followed by centrifugation at 10,000 rpm, 4 °C for 30 min.Screens of IEX media were performed on the Poly-Prep®Chromatography Columns (Bio-Rad, Hercules, CA, USA) packaged 2ml of different cation or anion resins listed in Table 1 as described previously . Note that, in case of cation resins, pHs of cell lysates disrupted with PBS buffer should be adjusted to pH 4.0 with acetic acid before centrifugation. After washing with 5 volumes of 20 mM acetate buffer pH 4.0/Tris-HCl buffer pH 8.0, bound proteins were eluted by 5 mL of PBS containing 1M NaCl, and elutions were analyzed by SDS-PAGE.
Purification of the PPV VLPs
To purify PPV VLPs by IEX chromatography, yeast cells were suspended in PBS buffer pH7.4 and disrupted by high-pressure homogenization. Cell lysates were subsequently adjusted to pH 4.0. After centrifugation at 10,000 rpm, 4 °C for 30 min, the supernatants of pH adjusted cell lysate were loaded onto an XK 50/30 column (GE Healthcare) packed with 400 ml of Capto S ImpAct resin. Binding VLPs were eluted with 20mM sodium acetate buffer containing 500 mM NaCl. To elevate the recovery of VLPs, the precipitates of pH adjusted cell lysates were redissolved in an equal volume of 20mM Tris-HCl buffer pH 8.0. Through centrifugation, the clarified supernatant were loaded onto an XK 50/30 column packed with 400 ml of Capto Q XP resins. After elution with 20mM Tris-HCl buffer pH 8.0 plus 500 mM NaCl, fractions were diafiltrated for 10 volumes of PBS on ÄKTA flux (GE Healthcare, USA) equipped with a 750 kDa column (11-0005-50, GE healthcare). Further polishing purification of PPV VLPs was performed on an AKTA Purifier 100 (GE Healthcare, USA) using a HiPrep™ 26/60 Sephacryl® S-500 HR column (GE Healthcare). About 4ml IEX purified sample was injected and eluted with PBS at a rate of 0.5ml/min. Protein concentration was measured by the BCA Protein Assay Kit (23250, Thermo Fisher Scientific).
Vaccination of mice with PPV VLPs
Purified VLPs were diluted with PBS buffer and emulsified with MONTANIDE™ Gel 01 adjuvant (Seppic, Paris, France) at a rate of 10 %, giving a final antigen concentration of 240 μg/ml. Fifteen of 6-week old female SPF Balb/c mice were randomly divided into 3 groups (n=5). Grouped mice were subcutaneously injected with 20 μg, 40μg PPV VLPs, and 250 μl of PBS as a control, respectively. Blood samples were collected from cheek each week till 49 dpi. Sera were separated by incubating blood at 37°C for 1 h, followed by centrifugation at 3,000 rpm for 4 min, and stored in small aliquots at -20°C.
Antibodies detection by enzyme-linked immunosorbent assay (ELISA)
96-well Costar Assay Plates (Corning, NewYork, USA) were coated with the Ni-NTA purified VP2 protein according to the method described previously . ELISA detections of the anti-PPV IgG titers in mouse sera were performed as described previously by Duan et al .
Serum hemagglutination inhibition (HI) antibody assay
HI antibody titers of serum samples were determined in U-bottom 96-well plates according to the standard method . Briefly, serum samples were inactivated at 56 °C for 30 min. Before the test, non-specific inhibitors of hemagglutinin in serum samples were removed by treating with 25 % kaolin and 3 % porcine erythrocytes. The treated sera were serially diluted 1:4 with PBS buffer, and each 25 μl of serum diluent was mixed with an equal volume of 40 μg/ml purified PPV VLPs. The plates were incubated for 1 h at 37 °C, and then 50 μL of 1% porcine erythrocyte was added per well. Finally, the plates were incubated at room temperature for 40 min to calculate the HI titers based on the reciprocal of the highest dilution inhibited hemagglutinin completely.
Spleen lymphocyte proliferation and Cytokine detection assay
At 42 dpi, three mice from the groups injected with 20 μg PPV VLPs and PBS were euthanized to separate spleen lymphocytes in a mouse lymphocyte separation medium (Dakewe, Beijing, China). The separated spleen cells were then cultivated in 96-well plates containing 100 μl of 1640 culture medium (Thermo Fisher Scientific, Illinois, USA), at a concentration of 3×106 cells/ml. After adding 0.2 μg Concanavalin A (Sigma, MO, USA), the plates were incubated at 37 °C for 48 h. Proliferative responses were detected using the Cell Titer 96® AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA), and stimulation indexes (SI) were calculated as the ratio of the stimulated sample divided to the unstimulated control at OD490 nm. Cytokines secreted by the spleen cells were measured using the Cytometric Bead Array (CBA) Mouse Th1/Th2 Cytokine Kit (Becton Dickinson Biosciences, San Jose, CA, USA).