Transformation of Salt Tolerant Lactuca sativa with Cholera toxin B Gene for Production of Edible Vaccine

Lettuce (Lactuca sativa) 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 study was conduct to express CTB gene in lettuce chloroplast. After surface sterilization of lettuce seeds, they were germinated on Murashige and Skoog (MS) half strength medium. Direct regeneration of lettuce by tissue culture was carried out in the full strength MS medium containing 0.1 mg/l alpha-Naphtaleneacetic acid (NAA) and 0.2 mg/l 6-benzylaminopurine (BAP). Lettuce plant was experimented for tolerance of betaine aldehyde (BA) and sodium chloride (NaCl) by tissue culture technique and it was found that the wild type lettuce tolerated 10 and 75 mM from each substance respectively. Genes required in this study were obtained by polymerase chain reaction (PCR) technique using specific forward and reverse primers, and these genes were cholera toxin B subunit (CTB), betaine aldehyde dehydrogenase (BADH),and prrn promoter and many other regulatory genes. Some of these genes were isolated from their hosts(Vibrio cholera, Spanish, and lettuce) 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. Transformation event was high when gene gun technology was used and it
was found that the distance of 6 cm and 900 pound per square inch (psi) were the optimum parameters to get high transformation frequency reaching 10% transformation events per bombarded leaf .In addition, more efficient age of leaves for bombardment was 40 days. The B subunits of toxin of Vibrio cholerae (CTB) are the candidate vaccine antigens. In this work the CTB gene with BADH gene were transferred to the chloroplast of lettuce plant and selection of transformed plant cell 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. 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 trnaI\trnA.Western blot analysis showed that the chloroplast synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Transgenic lettuce plant showed ability to tolerate 150 mM NaCl and 35 mM BA. Introduced genes were stable and inherited in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their functions, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.