Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic rice (Oryza sativa L.)

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as a potent mucosal immunogen and immunoadjuvant for co-administered antigens, The synthetic LTB (sLTB) was modified based on plant optimized codon usage, and fused to a translation signal (the Kozak sequence) in the front of start codon and the ER retention signal, SEKDEL, in the c-terminus of sLTB gene. The sLTB and the wild-type LTB gene (wLTB) were located into plant expression vectors under the control of the wheat B×17 HMW (High Molecular Weight) glutenin endosperm-specific promoter containing the first intron of the rice actin1 gene. Both genes were introduced into dce cells (Oryza sativa L.) via particle bombardment mediated transformation. The integration of LTB gene into the chromosome of transgenic plants was confirmed by genomic DNA PCR amplification methods. The transcription and translation of the LTB genes were demonstrated by reverse-transcription PCR (RT-PCR) and Western blot analyses, respectively. The LTB proteins produced in the seed tissues of transgenic rice showed binding affinity for G_m1 ganglioside, a receptor for biologically active LTB, suggesting the plant-produced LTB are capable of forming active pentamers. The expression level of sLTB was higher than wLTB in transgenic rice plants and was up to 2.7% of the total soluble proteins of the seed tissues.