A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice

Figure 1 Transplastomy of the spectinomycin-resistant plants. A: Maps of WT and transplastomic genomes in the area of two arms (psaB-trnfM and trnG-psbC) for homologous recombination. Horizontal arrows indicate locations and orientations of different primers. The sizes of PCR products are shown in kb. Arrows pointing upwards denote nucleotide positions in the tobacco chloroplast genome (TCG) in accordance with GenBank accession no. Z00044. PRpsbA: rice psbA promoter; TRpsbA: rice psbA terminator; PTrrn: tobacco rrn promoter; TTpsbA: tobacco psbA terminator. B: Agarose gel pictures of PCR products. Primers used for generating each product are given to the right of the panels. Arrows indicate sizes of the PCR products in kb. Primer pair Hel-19/Hel-24 amplified a 1.4-kb fragment in the rbcL gene presenting in both the WT and the transplastomic plants.

Figure 2 Transformed plastid genomes revealed by Southern blot analysis. A: Maps of WT and transplastomic genomes showing the positions of KpnI and NcoI sites, and areas to which different probes hybridize. The restriction fragment lengths are given in kb. B: DNA gel blot analysis confirming transgene integration into chloroplast genomes. DNA was digested by KpnI and NcoI. The blots were hybridized with probes targeting the psaB-trnfM arm (P1), trnG-psbC arm (P2), rice psbA promoter (P3) and aadA-coding region (P4). Arrows indicate the sizes of the hybridized DNA fragments in kb.

Figure 3 Transgene expression investigated by Northern blot analysis. A: Map of the transplastomic genome with areas to which different probes hybridize. Lengths of possible transcripts are shown in kb. B: RNA blot analysis for characterization of different transcripts. RNA blots were hybridized with probes targeting the aadA-coding region (P4), HEV pE2-coding sequence (P5), junction between the rice psbA terminator and tobacco rrn promoter (P6) and tobacco trnfM (P7). Arrows to the left of the panels denote lengths of RNA in kb. Normalization of RNA loading was demonstrated by ethidium bromide staining of the RNA gel.