Enhanced expression of dengue virus EDIII-based tetravalent antigen protein using transgenic rice callus

Mosquito-borne tropical dengue disease is a major global epidemic affecting hundreds of millions of people. As a precaution, it would be ideal to develop a highly efficient tetravalent vaccine that protects against all serotypes of dengue viruses. In this study, envelope protein domain III (EDIII) of dengue virus was employed as a target antigen. A single-chain tetravalent type of EDIII (tEDIII) was generated by utilizing a linker peptide and transformed into rice (Oryza sativa). Integration and mRNA transcripts of the tEDIII gene were confirmed by genomic DNA polymerase chain reaction (PCR) and Northern blot analyses, respectively. The expression of wild-type tEDIII (wtEDIII) protein was confirmed by Western blot analysis and was determined to improve the synthesis of the tEDIII (stEDIII) construct based on codon optimization and ER targeting. The yield of stEDIII protein was sevenfold higher than secreted wtEDIII protein, reaching a maximum of 357 µg per gram dry weight. These results suggest that a simple tetravalent EDIII dengue antigen can be produced in rice, raising the possibility that edible plant cells can be vaccinated by mucosal application for protection against dengue infection.