Figure 1.
Schematic diagram of the pEAQ-HT-HA plant expression system construct used for agro-infiltration.
The entire T-DNA region is shown (not to scale): Black boxes, T-DNA borders (RB, right border; LB, left border); white arrows, CaMV 35S promoters; grey arrows, open reading frames; black arrows, Nos terminators; and NPTII, neomycin phosphotransferase II gene.
Table 1.
Rabbit antisera raised against influenza A virus subtypes H1-H12.
Figure 2.
Phylogenetic relationships between the A/mallard/Sweden/7206/2004(H7N7) isolate and representative H7 sequences available in public databases.
Bayesian phylogeny of HA amino acid sequences (denoted by GenBank accession number and isolated description) was inferred using the MrBayes 3.1.2 program and the Jones model of substation with gamma-distributed rate variation across sites. Nodes with posterior probability support ≥95% are indicated. The Swedish H7 sequence isolated is highlighted in bold face. Scale bar indicates the number of amino acid changes per branch length.
Figure 3.
Relative expression of plant-produced recombinant hemagglutinin was measured by quantitative real-time PCR.
The expression levels was measured at 3, 6, 9, 12 and 15 dpi (days post infiltration) and are normalized to expression of control genes, the ubiquitin (ubi3) and elongation factor-1 (EF-1). The results represent two separate experiments, each time performed including three technical replicates.
Figure 4.
Plant-produced recombinant full-length hemagglutinin (rHA0) analysis using SDS-PAGE and western blot.
A: Probe with anti-His antibodies, Lane 1, molecular weight marker; lane 2, purified rHA0 (5 µg) from total soluble protein (TSP) at 6 days post infiltration (dpi) (SDS-PAGE); lane 3, TSP (40 µg) from untreated Nicotiana benthamiana leaves (negative control; 0 dpi); lanes 4 to 8, TSP (40 µg protein per lane) from the agro-infiltrated leaves of N. benthamiana at 3, 6, 9, 12 and 15 dpi; lane 9, purified rHA0 (5 µg) at 6 dpi; lane 10, purified rHA0 (5 µg) at 6 dpi after PNGase F digestion. The shift to a lower molecular mass upon treatment with PNGase F is indicative of N-linked glycosylation on rHA0; B: Probe with anti-HA antibody, Lane 1, molecular weight marker, lane 2, purified rHA0 (5 µg) at 6 dpi.
Figure 5.
Triplicates of purified plant-produced hemagglutinin (rHA0) were two-fold serially diluted beginning with a concentration of 10 µg/mL from the stock of 400 µg/mL and mixed with chicken erythrocytes. Wells in the bottom row contains bovine serum albumin (BSA) as a negative control starting with a concentration of 10 µg/mL from 400 µg/mL stock. The lowest concentration (µg/mL) of the well showing complete agglutinating activity of erythrocytes was considered as hemagglutination titers (HT).
Figure 6.
Hemagglutination inhibition assay.
For the assay, 4 HAU of purified plant-produced recombinant hemagglutinin (rHA0) were mixed with 2-fold serial diluted hyperimmune rabbit antiserum (starting at a dilution of 1∶80) generated against various subtypes of viral strains or phosphate buffer saline (PBS) (negative control). Native Swedish H7N7 influenza A virus isolate was used as positive control. Formation of immune complexes was allowed for 30 min at 37°C, before incubation with chicken erythrocytes for 1 h at 4°C.