by: abigail skorupski. background info what is cholera? vibrio cholerae symptoms cholera toxin a...
TRANSCRIPT
![Page 1: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/1.jpg)
A Potato a Day Keeps the Cholera Away
By: Abigail Skorupski
![Page 2: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/2.jpg)
Background Info What is cholera?
Vibrio Cholerae
Symptoms Cholera Toxin
A subunitB subunit
Vaccination
Countries Affected by Cholera
Vibrio Cholerae
![Page 3: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/3.jpg)
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
![Page 4: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/4.jpg)
Transformation of Potato PlantsLeaf explants placed in dish w/AgrobacteriumTransformed plant cells moved to selective
mediumShoot and root formationPlantlets grown into mature plants
![Page 5: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/5.jpg)
Luciferase Activity#1-6 = Kanamycin
resistant plants#7-12 =
untransformed potato plants
A&B = in duplicateC&D = in duplicate
Plants #1, 3, 4 chosen
![Page 6: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/6.jpg)
Analysis of CTB-SEKDEL fusion gene
PCR amplification
Gel electrophoresis
Plant #4 chosen for Immunoblot analysis
![Page 7: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/7.jpg)
Immunoblot AnalysisNon-fat dry milkRabbit anti-cholera
antiserumMouse anti-rabbit
IgG conjugated to alkaline phosphatase
Chemiluminescent substrate CSPD
![Page 8: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/8.jpg)
Quantification of CTB proteinELISA assaysBackground blocked
by BSARxn measured in
plate luminometer0.3% = max amt.1 g = 30-35 µg
recombinant CTB protein
![Page 9: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/9.jpg)
Oral Immunization of CD-1 Mice30 µg bacterial CTB1 g transformed potato tissue3 g transformed potato tissuechemiluminescent ELISANeutralization titers
![Page 10: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/10.jpg)
Reduction of CT-Induced Diarrhea
Immunological protection due to prevention of binding to cellular GM1-ganglioside
![Page 11: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/11.jpg)
Human Clinical Trials#TH110-51
potatoes14 volunteers:
100 g transgenic
50 g transgenic50 g wildtype
![Page 12: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/12.jpg)
• 91% = 4-fold rises in IgG anti-LT• 73% = neutralization titers > 1:100• 50% = 4-fold rises in sIgA
![Page 13: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/13.jpg)
Issues with Transgenic PlantsLevel of expression of
foreign gene can vary greatly
Oral toleranceGreenpeace
![Page 14: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/14.jpg)
ConclusionsEdible vaccines = safe and inexpensiveBananas as vaccines
![Page 15: By: Abigail Skorupski. Background Info What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera](https://reader033.vdocuments.site/reader033/viewer/2022061612/56649db35503460f94aa3d44/html5/thumbnails/15.jpg)
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.