- All Journals
- Al-Qadisiyah University
- Basrah University
- Kirkuk University
- Mosul University
- The Iraqi Borad for Medical Specialization
- University of Anbar
- University of Technology
- Al Mustafa University
- Al-Zahraa University for Women
- Alnoor University
- Kunooz University
- Misan University
- Ninevah University
- Tikrit University
- University of Al-Hamdaniya
- University of Kerbala
Production of Lettuce Edible Vaccine for Cholera Disease Using Chloroplast Genetic Engineering. | ||
| Journal of University of Anbar for Pure Science | ||
| Article 7, Volume 7, Issue 1, 2013, Pages 20-27 PDF (610.94 K) | ||
| Document Type: Research Paper | ||
| DOI: 10.37652/juaps.2013.83114 | ||
| Authors | ||
| Ahmed A. Suleiman* 1; Kadhim M. Ibrahim2; Abdulwahid B. Al-Shaibani2 | ||
| 1University Of Alanbar - College of women Education | ||
| 2Nahrin University College of Science. | ||
| 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 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 | ||
| Keywords | ||
| Cholerae; edible vaccine; choloplast genetic engineering; CTB | ||
| References | ||
|
1- Ruhlman, T.; Verma, D.; Samson, N. and Daniell, H.(2010). The role of heterologous chloroplast sequence elements in transgene integration andexpression. Plant phys., 152:2088-2100.
2-Maliga, P. and Bock, R.(2011). Plastid Biotechnology: Food, Fuel, and Medicine for the21st Century. Plant Physiol., 155: 1501–1510.
3- Ruhlman, T.; Ahangari, R.; Devine, A.; Samsam, M. and Daniell, H. (2007). Expression of cholera toxin B-proinsulin fusion protein in lettuce andTobacco chloroplasts—oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnology J., 5: 495-510.
4- Daniell, H.; Lee, S.B; Tanvi, P. and Peter O. W.(2001). Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol., 311: 1001-1009.
5- Verma D. and Daniell H. (2007). Chloroplast vector systems forbiotechnology applications. Plant Physiol., 145: 1129-1143.
6- Verma, D.; Samson, N.P.; Koya, V. and Daniell, H. (2008). A protocol for expression of foreign genes in chloroplasts. Nat. Protoc., 3: 739-758.
7- Sambrook, J.; Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning; ALaboratory Manual. Cold Spring Harbor Laboratory Press, New York, NY.
8- Kumar, S.; Dhingra, A. and Daniell, H.(2004). Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance. Plant Physiology, 136: 2843–2854.
9- Singh, N.D.; Ding, Y. and Daniell, H.(2009). Chloroplast-derived vaccineantigens and biopharmaceuticals: Protocols for expression, purification, or oral delivery and functional evaluation. In: Methods in Molecular Biology, Recombinant Proteins From Plants, vol. 483:163-210.
10- Jani, D.; Laxman Singh, M.; Quazi Mohammad, R.H.; Yogendra, S.;vArun, K. S. and Akhilesh, K. T.(2002). Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Research, 11:447-454.
11- Foolad, M.R.(2004). Recent advances in genetics of salt tolerance in tomato. Plant Cell, 76: 101–119.
12- De Cosa, B.; Moar, W.; Lee, S.B.; Miller, M. and Daniell, H. (2001). Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat. Biotechnol.,19: 71-74.
13- Daniell, H. ; Singh, N.D.; Mason, H. and Streatfield, J. (2010). Plant-made vaccine antigens and Biopharmaceuticals. Trends in plant science, 13:488-489.
14- DeGray, G.; Rajasekaran, K.; Smith, F.; Sanford, J. and Daniell, H. (2001). Expression of an antimicrobial peptide via the chloroplast genome to control phytopathogenic bacteria and fungi. Plant Physiol., 127:852– 862. folding and assembly of the CTB pentamer 4. We here expressed CTB in plastids of lettuce using BADH gene for salt tolerance as selectable marker gene. | ||
|
Statistics Article View: 423 PDF Download: 335 |
||