Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato.

A CTB-NSP4(175) fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB-NSP4(175) enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification. Synthesis and assembly of the full-length CTB-NSP4(175) fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis. The binding of CTB-NSP4(175 )fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). The ELISA results showed that CTB-NSP4(175) fusion protein was 0.006-0.026% of the total soluble tuber protein. The synthesis of CTB-NSP4(175) monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response.