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A Potato a Day Keeps the Cholera Away
By: Abigail Skorupski
Background Info What is cholera?
Vibrio Cholerae
Symptoms Cholera Toxin
A subunitB subunit
Vaccination
Countries Affected by Cholera
Vibrio Cholerae
Construction of Plant Expression VectorPpcv701FM4-CTB:SEKDEL
-CTB gene fused with ER retention signal SEKDEL
-Bacterial luciferase AB fusion gene
-NPT-II expression cassette
-β-lactamase cassette-Removed Shine-Delgarno
Transformation of Potato PlantsLeaf explants placed in dish w/AgrobacteriumTransformed plant cells moved to selective
mediumShoot and root formationPlantlets grown into mature plants
Luciferase Activity#1-6 = Kanamycin
resistant plants#7-12 =
untransformed potato plants
A&B = in duplicateC&D = in duplicate
Plants #1, 3, 4 chosen
Analysis of CTB-SEKDEL fusion gene
PCR amplification
Gel electrophoresis
Plant #4 chosen for Immunoblot analysis
Immunoblot AnalysisNon-fat dry milkRabbit anti-cholera
antiserumMouse anti-rabbit
IgG conjugated to alkaline phosphatase
Chemiluminescent substrate CSPD
Quantification of CTB proteinELISA assaysBackground blocked
by BSARxn measured in
plate luminometer0.3% = max amt.1 g = 30-35 µg
recombinant CTB protein
Oral Immunization of CD-1 Mice30 µg bacterial CTB1 g transformed potato tissue3 g transformed potato tissuechemiluminescent ELISANeutralization titers
Reduction of CT-Induced Diarrhea
Immunological protection due to prevention of binding to cellular GM1-ganglioside
Human Clinical Trials#TH110-51
potatoes14 volunteers:
100 g transgenic
50 g transgenic50 g wildtype
• 91% = 4-fold rises in IgG anti-LT• 73% = neutralization titers > 1:100• 50% = 4-fold rises in sIgA
Issues with Transgenic PlantsLevel of expression of
foreign gene can vary greatly
Oral toleranceGreenpeace
ConclusionsEdible vaccines = safe and inexpensiveBananas as vaccines
References Arakawa T, Chong D, Langridge W. Efficacy of a food plant-based oral
cholera toxin B subunit vaccine. Nature Biotechnology [serial online]. March 1998;16(3):292-297. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012.
Arakawa T, Chong D, Merritt J, Langridge W. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Research
[serial online]. November 1997;6(6):403-413. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012.
Tacket C, Mason H. A review of oral vaccination with transgenic vegetables. Microbes And Infection / Institut Pasteur [serial online]. August 1999;1(10):777-783. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012.
Tacket C, Mason H, Losonsky G, Clements J, Levine M, Arntzen C. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Medicine [serial online]. May 1998;4(5):607-609. Available from: MEDLINE with Full Text, Ipswich, MA. Accessed December 2, 2012.