Production of Lettuce Edible Vaccine for Cholera Disease Using Chloroplast Genetic Engineering.

Lettuce is one of the most important edible plant worldwide. At the time that lettuce is the candidate plant to carry the foreign vaccine gene for human. The B subunits of toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. This research was conduct to express CTB gene in lettuce chloroplast. Genes required in this study were obtained by polymerase chain reaction (PCR) technique using specific forward and reverse primers, and these genes were CTB, BADH, prrn promoter and many other regulatory genes. Some of these genes were isolated from their hosts and some were obtained from previous work available at Daniell laboratory. All these genes beside many techniques for ligation, extension, sequencing, orientation confirmation were used to construct the cassette vector pLS-BADH-LS-CTB which carries the gene of interest. In this work the CTB gene with BADH gene were transferred to the chloroplast of lettuce plant and selection of transgenic plant was performed on the MS medium containing BA and NaCl without any antibiotic selectable marker. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 1000 copies per cell) resulted in the accumulation of up to 6.2% of total soluble lettuce leaves protein as functional oligomers (620-fold higher expression levels than that of the unmodified CTB gene expressed via the nuclear genome). PCR and Southern blot analyses confirmed stable integration of the CTB gene and BADH gene into the chloroplast genome in addition to the integration in the right orientation and in specific region between trnaItrnA.Western blot analysis showed that the chloroplast synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB.