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A Potato a Day Keeps the Cholera Away

A Potato a Day Keeps the Cholera Away. By: Abigail Skorupski. Background Info. What is cholera? Vibrio Cholerae Symptoms Cholera Toxin A subunit B subunit Vaccination. Countries Affected by Cholera. Vibrio Cholerae. Construction of Plant Expression Vector Ppcv701FM4-CTB:SEKDEL.

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A Potato a Day Keeps the Cholera Away

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  1. A Potato a Day Keeps the Cholera Away By: Abigail Skorupski

  2. Background Info What is cholera? VibrioCholerae Symptoms Cholera Toxin A subunit B subunit Vaccination Countries Affected by Cholera VibrioCholerae

  3. 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

  4. Transformation of Potato Plants • Leaf explants placed in dish w/Agrobacterium • Transformed plant cells moved to selective medium • Shoot and root formation • Plantlets grown into mature plants

  5. Luciferase Activity #1-6 = Kanamycin resistant plants #7-12 = untransformed potato plants A&B = in duplicate C&D = in duplicate Plants #1, 3, 4 chosen

  6. Analysis of CTB-SEKDEL fusion gene • PCR amplification • Gel electrophoresis • Plant #4 chosen for Immunoblot analysis

  7. Immunoblot Analysis • Non-fat dry milk • Rabbit anti-cholera antiserum • Mouse anti-rabbit IgG conjugated to alkaline phosphatase • Chemiluminescent substrate CSPD

  8. Quantification of CTB protein • ELISA assays • Background blocked by BSA • Rxn measured in plate luminometer • 0.3% = max amt. • 1 g = 30-35 µg recombinant CTB protein

  9. Oral Immunization of CD-1 Mice • 30 µg bacterial CTB • 1 g transformed potato tissue • 3 g transformed potato tissue • chemiluminescent ELISA • Neutralization titers

  10. Reduction of CT-Induced Diarrhea Immunological protection due to prevention of binding to cellular GM1-ganglioside

  11. Human Clinical Trials • #TH110-51 potatoes • 14 volunteers: • 100 g transgenic • 50 g transgenic • 50 g wildtype

  12. 91% = 4-fold rises in IgG anti-LT • 73% = neutralization titers > 1:100 • 50% = 4-fold rises in sIgA

  13. Issues with Transgenic Plants • Level of expression of foreign gene can vary greatly • Oral tolerance • Greenpeace

  14. Conclusions • Edible vaccines = safe and inexpensive • Bananas as vaccines

  15. 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.

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