Figure 2
Strategy to create a single tetravalent P. pastoris clone. (a) Plasmid pT harboring four DENV E ECs. Each EC consists of the AOX1 promoter (P), E gene of a single DENV serotype (E1: magenta, E2: green, E3: blue, E4: black) and the AOX1 terminator (T). ‘B/B’ denotes the Bam HI/Bgl II fusion site created by the ligation of the 3′ end of one EC to the 5′ end of the next in the tandem array. The plasmid contains 3′ AOX1 sequences and HIS4 marker for integration and selection in P. pastoris, respectively, as well as bacterial sequences for replication (Ori) and ampicillin selection (AmpR) in E. coli. (b) Agarose gel analysis of PCR products obtained using plasmid pT (lanes ‘T’) as well as monovalent plasmids containing E gene of DENV-1 (lane 1), DENV-2 (lane 2), DENV-3 (lane 3) and DENV-4 (lane 4), as templates, in conjunction with primer pairs P1, P2, P3 and P420, specific to genes E1, E2, E3 and E4, respectively. (c) Agarose gel analysis of restriction digests of plasmid pT performed with Bgl II + Bam HI (B + B), Afl II and Pst I. (d) PCR analysis of P. pastoris genomic DNA. Genomic DNA from the tetravalent P. pastoris clone (T) and from untransformed P. pastoris host (N, negative control) were analysed by PCR for the presence of E1 (lanes ‘1’), E2 (lanes ‘2’), E3 (lanes ‘3’) and E4 (lanes ‘4’) genes, using the same gene-specific primer pairs as in panel ‘b’. Monovalent (Mono) E1, E2, E3 and E4 plasmid DNAs were analysed in parallel as positive controls (in ‘Mono’ lanes 1–4). Panels ‘b’ and ‘d’ represent full gels; the gel in panel ‘c’ is cropped to eliminate lanes in which unrelated samples were run (the full gel picture is included in supplementary file, page 5. For panels ‘b-d’, DNA size markers were analysed in lanes ‘M’. Their sizes (in kb) are shown to the left of each panel. Predicted sizes of PCR amplicons (panels ‘b’,‘d’), location of restriction sites and the expected restriction fragments (panel ‘c’) are indicated in Supplementary Fig. S2.
