Expression of a Cholera Toxin B Subunit-Rotavirus Enterotoxin Fusion Gene in Transgenic Potato
Tae-Geum Kim, Nak-Won Choi, William H.R. Langridge
To increase the number of available epitopes in plant subunit vaccines for enhancement of the immune response to rotavirus infection, a fusion gene encoding the cholera toxin B subunit linked to a murine rotavirus enterotoxin 90 aa protein (CTB-NSP490) was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP490 fusion gene was detected in the genomic DNA of transformed plants by PCR analysis methods. Immunoblot analysis of transformed tuber extracts showed that synthesis and assembly of the CTBNSP490 fusion protein into pentameric size oligomers was occurred in the plant cells. The binding of CTB-NSP490 fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). The ELISA results indicated that the CTB-NSP490 fusion protein was synthesized at 0.071 to 0.18% of total soluble tuber protein. Synthesis of CTB-NSP490 monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using potato tubers as a production and delivery system for enterocyte targeted rotavirus enterotoxin proteins of a size large enough to provide the maximum number of pathogenic epitopes for stimulating the strongest possible mucosal immune response.