Figure 3

Analysis of co-expression of E mRNAs and proteins. (a) Real time analysis of DENV E1, E2, E3 and E4 mRNA co-expression in methanol-induced P. pastoris transformed with plasmid pT. Total RNA was isolated from the methanol-induced tetravalent clone of P. pastoris, transcribed to cDNA using reverse primers specific to E1 (curve 1), E2 (curve 2), E3 (curve 3) or E4 (curve 4) and subjected to qPCR. Total RNA from P. pastoris transformed with the empty expression vector pAO815 was subjected to RT-qPCR in parallel as a negative control (curve N). (b) Similar experiment as in panel ‘a’, except that mRNAs for real-time analysis were obtained from methanol-induced monovalent P. pastoris clones harboring E1 (curve 1), E2 (curve 2), E3 (curve 3) or E4 (curve 4) gene. (c) Analysis of co-expression of all four DENV E proteins with serotype-specific mAbs using indirect ELISA. Microtitre wells were coated either with methanol-induced P. pastoris lysate obtained from the tetravalent ‘4-in-1’ clone (orange bars) or purified E proteins obtained from the four different monovalent P. pastoris clones: E1 (magenta bar), E2 (green bar), E3 (blue bar) or E4 (black bar) proteins, and probed with mAbs specific to E proteins of DENV-1 [E29 (1)], DENV-2 [3H5 (2)], DENV-3 [E1 (3)] or DENV-4 [E42 (4)]. Bound mAbs were detected using secondary anti-mouse IgG-HRPO conjugate. Data shown are the mean of triplicates (error bars denote standard deviation, SD). Each experiment was performed at least twice independently with data from one typical experiment shown in each of the three panels.