Preparation of a tetravalent ED3-expressing DNA vaccine and a tetravalent recombinant ED3 protein vaccine.

<p>(A) Schematic diagram of two ED3-expressing plasmids, pDV13-ED3 and pDV24-ED3, generated by cloning DNA fragments consisting of tandem repeats of ED3 from DENV-1 and 3, and DENV-2 and 4 into the pVax-1 vector between the Xho I and Apa I sites. The positions of the immunoglobulin light chain signal peptide (Ig) or three repeats of GGGGS linker (linker) are also indicated. (B) The presence of bivalent ED3 in the culture supernatants or cell lysates of 293Trex cells transfected with pVax-1, pDV13-ED3 or pDV24-ED3 for 24 h was detected with an ED3-specific monoclonal antibody. The expression of β-actin cellular protein in the cell lysate was used as a control. The recombinant ED3 proteins from DENV-1 to 4 were expressed in <i>E</i>. <i>coli</i> and purified with an affinity column. The final products were analyzed by SDS-PAGE (C) or Western blotting (D) with hyperimmune sera.