Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297–394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB–EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB–EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB–EIII fusion protein in transgenic plant extracts. Based on the GM1-ELISA results, the CTB–EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, GM1-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB–EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.