Ahmed A. Suleiman; Kadhim M. Ibrahim; Abdulwahid B. Al-Shaibani
Abstract
Lettuce is one of the most important edible plant worldwide. At the timethat lettuce isthe candidate plant to carry the foreign vaccine gene forhuman. The B subunits of toxin of Vibrio ...
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Lettuce is one of the most important edible plant worldwide. At the timethat lettuce isthe candidate plant to carry the foreign vaccine gene forhuman. The B subunits of toxin of Vibrio cholerae(CTB) are candidatevaccine antigens. This research was conduct to express CTB gene in lettucechloroplast. Genesrequired in this study were obtained by polymerase chain reaction (PCR)technique using specific forward and reverse primers, and these genes wereCTB, BADH, prrnpromoter and many other regulatory genes. Some ofthese genes were isolated from their hosts and some were obtained fromprevious work available at Daniell laboratory. All these genes beside manytechniques for ligation, extension, sequencing, orientation confirmationwere used to construct the cassette vector pLS-BADH-LS-CTB whichcarries the gene of interest. In this work the CTB gene with BADH genewere transferred to the chloroplast of lettuce plant and selection oftransgenic plant was performed on the MS medium containing BA andNaCl without any antibiotic selectable marker. Integration of an unmodifiedCTB-coding sequence into chloroplast genomes (up to 1000 copies per cell)resulted in the accumulation of up to 6.2% of total soluble lettuce leavesprotein as functional oligomers (620-fold higher expression levels than thatof the unmodified CTB gene expressed via the nuclear genome). PCR andSouthern blot analyses confirmed stable integration of the CTB gene andBADH gene into the chloroplast genome in addition to the integration in theright orientation and in specific region between trnaI rnA.Western blotanalysis showed that the chloroplast synthesized CTB assembled intooligomers and were antigenically identical with purified native CTB