Expression of a cholera toxin B subunit and consensus dengue virus envelope protein domain III fusion gene in transgenic rice callus

Dengue (DEN) is one of the most important emerging mosquito-borne viral human diseases. Therefore, an effective dengue vaccine with immune responses against all four dengue virus serotypes is highly needed. A fusion gene encoding a synthetic consensus envelope protein domain III (scEDIII) of dengue virus with neutralizing activity against the four dengue virus serotypes and with the B subunit of cholera toxin (CTB) to increase its mucosal immunogenicity was constructed and was introduced into rice callus under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of the CTB-scEDIII fusion gene in transgenic rice callus were confirmed by genomic DNA PCR amplification, Northern, and Western blot analyses, respectively. The biological binding activity of the CTB-cEDIII fusion protein to its G M1 -ganglioside receptor was confirmed via G M1 -ELISA with anti-CT and anti-dengue virus antibodies. Delivery of the CTB-cEDIII fusion protein into mucosal immune inductive sites (including M cells) in BALB/c mice was confirmed by in vitro and in vivo antigen uptake assays. These results showed that the CTB-cEDIII fusion protein was produced in the transgenic rice callus, and that plant-produced ligand fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of the overall immune responses.