Figure 1.
Pichia pastoris expression cassette in pPICZ_B (Invitrogen).
Abbreviations: 5′AOX1 and AOX1 TT, methanol-inducible alcohol oxidase 1 gene promoter and terminator, respectively; CHS 5′-UT, untranslated region of the Petroselinum hortense chalcone synthase gene; IBD-VP2, cDNA of Infectious bursal disease virus protein 2, corresponding to the first 441 amino acids; H6, His-6 tag for detection and purification; pTEF1, transcription elongation factor 1 gene promoter from S. cerevisiae that drives expression of the Sh ble gene in P. pastoris conferring zeocin resistance; pEM7, constitutive synthetic prokaryotic promoter that drives expression of the Sh ble gene in E. coli; Sh ble, Streptoalloteichus hindustanus bleomycin resistance gene; Cyc1 TT, CYC1 transcription termination region (GenBank accession number M34014), the 3′ end of the S. cerevisiae CYC1 gene that allows efficient 3′ mRNA processing of the Sh ble gene for increased stability.
Table 1.
Experimental design for chicken immunization.
Figure 2.
Characterization of recombinant IBD-VP2 produced in Pichia pastoris.
(A) A 12-µl aliquot of the P. pastoris cell lysate was separated by SDS-PAGE (12% (w/v) polyacrylamide) and blotted onto a nitrocellulose membrane. The antigen was detected using a rabbit anti-IBD-VP2 (1∶10,000) primary antibody and an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (0.12 µg/ml). The signal was detected with NBT/BCIP for 5 min at room temperature. (B) Intracellular accumulation level of IBD-VP2 in P. pastoris cultures following expression induction. Biological triplicates were used for each experiment.
Figure 3.
Separation of partially-purified yeast-derived IBD-VP2 by SEC and analysis by electron microscopy studies.
(A) The IBD-VP2 protein was produced and extracted from the P. pastoris cells using breaking buffer (pH 4.0) followed by precipitation with 50% ammonium sulfate. The protein pellet was resuspended in PBS and separated by SEC using a S-400 HR column with a size separation range 20–8000 kDa. (B) The eluted fractions were tested for IBD-VP2 content using an indirect ELISA, revealing IBD-VP2 peaks at 152 and 250 ml during SEC. Electron microscopy revealed that only the 152-ml peak contained fully-assembled 23-nm IBD-SVPs. Scale bar = 50 nm.
Figure 4.
Analysis of IBD-SVP stability and integrity after yeast cell inactivation using SDS-PAGE and SEC.
We separated 12-µl samples by SDS-PAGE (12% (v/w) polyacrylamide) followed by staining with Coomassie Brilliant Blue (A) and immunoblot detection (B) using a rabbit anti-VP2 primary antibody, an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (0.2 µg/ml) and NBT/BCIP to detect the signal: (1) purified IBD-SVPs derived from freeze-dried inactivated P. pastoris cells; (2) purified IBD-SVPs derived from active freeze-dried P. pastoris cells; (3) purified IBD-SVPs as a positive control. (C) Chromatogram obtained by separation of the corresponding samples using a SEC S-400 HR column and Äkta Explorer.
Figure 5.
Immunization of chickens with IBD-VP2 or purified IBD-SVPs produced in P. pastoris.
(A) Immunization scheme: two-week-old chickens were immunized at the indicated times (schematized as black bars) by either oral (groups 1–9 and 12) or intramuscular (group 10) administration. Oral immunization was carried out using different doses of either freeze-dried yeast (groups 1–4) or purified yeast-derived IBD-SVPs (groups 5–8) with and without an oral adjuvant mixture. Wild-type P. pastoris X-33 cells were used as a negative control (group 9) and Avipro Gumboro vac was used as a positive control (group 12). Intramascular immunization was carried out using 20 µg of purified yeast-derived IBD-SVPs mixed with Adjuvant 100 (Gerbu). Two groups remained unvaccinated, one in the challenge experiment and used as a challenge control (group 11) and the other unchallenged as a vaccine control (group 13). The gray bar indicates the time interval following viral challenge; arrows indicate the time of death. The reactivity of chicken sera IgM (B) and IgY (C) with IBD-SVPs was determined by ELISA. The serum samples were collected before immunization, before viral challenge and one week after challenge. The mean of absorbance at 405 nm is shown for five chickens in each group with standard error indicated by error bars. The 0.2 OD value was used as a cut off; chickens with a higher antibody titer were considered seropositive.
Table 2.
Analysis of the antibody response in immunized chickens before and after challenge with IBDV.
Table 3.
Summary of the results obtained from immunized chickens challenged with IBDV.