1. Expression of Cholera toxin B subunit in Banana callus culture Dawn Rivard

2. Cholera An infection of the small intestine caused by the bacterium Vibrio cholerae ◦ A gram negative comma-shaped bacterium with a polar flagellum The main symptoms are profuse watery diarrhea, vomiting and abdominal pain Transmission is primarily through contaminated drinking water or food Can lead to dehydration and electrolyte imbalance

3. Cholera toxin An oligomeric complex made up of 6 protein subunits ◦ 1 copy of the A subunit (enzymatic) ◦ 5 copies of the B subunit (receptor binding) ◦ Connected by a disulfide bond The B subunits form a five-membered ring and part A forms an extended alpha helix which fits in the central pore of the ring

4. Mechanism of action The pentameric part B of the toxin molecule binds to the surface of the intestinal epithelium cells Part A detaches from part B upon binding and gets inside the cell via receptor-mediated endocytosis

5. Mechanism of action Part A permanently ribosylates the Gs alpha subunit of the heterotrimetric G protein resulting in constitutive cAMP production This leads to secretion of H ₂O, Na⁺, K⁺, Cl⁻, and HCO₃⁻ into the lumen of the small intestine

6. Experiment In this study, an attempt was made towards the production of edible vaccine by expressing CT-B subunits of cholera toxin in Banana callus culture, through Agrobacterium mediated gene transfer methods.

7. Experiment The CT-B antigen was prepared from Vibrio cholerae. The size of the antigen was confirmed by SDS- PAGE. The antigen was eluted from SDS- PAGE and then used for vector construction.

8. Cloning vector E. coli DH5 α PRK2013 together with pBluescript II KS were used for the initial cloning, sequencing and maintenance of different DNA fragments.

9. Cloning vector DNA fragments were analyzed by electrophoresis on an agarose gel and purified. Oligonucleotide primers were designed according to the published sequence for CT-B. PCR was carried out to create BamHI-EcoRI CT-B cloning cassette. The amplified CT-B cassette was confimed by digesting with both BamHI and EcoRI and recovered in pBluescript KS II. PCR fidelity was verified by complete sequencing of the CT-B portion.

10. Expression vector The cassette was excised and sub cloned between BamHI and EcoRI sites of PGA 643 to create plant expressing plasmid pCAMBIA. CT-B gene was amplified by PCR and cloned into a vector containing the strong, constitutive 35S CaMV promoter and a reiterated 35S enhancer. The plasmids were transformed into Banana callus via Agrobacterium tumefaciens.

11. Expression vector

12. Results The CT-B encoded protein was injected into a 3 months old callus of banana species by micro syringe method. The callus was maintained in the same culture chamber under aseptic conditions provided with light intensity and temperature control.

13. Results The subculture was maintained at regular intervals until able to differentiate the callus into plantlets. The remaining callus was allowed to grow in the same experimental conditions to differentiate into multiple shoots.

14. Results Integration of the transgene was confirmed by PCR using genomic DNA isolated from transformed and control cells. The recovered plasmid was further analyzed by PCR to confirm the presence of the CT-B cassette in the recovered plasmid by agarose gel electrophoresis.

15. Results The total soluble proteins were extracted from 1g of callus tissue. This was concentrated to 100µL by freeze drying in low speed in a high vacuum. 20µL of this was used for western blot analysis. Western analysis confirmed the presence of CT-B antigen specific band.

16. Results Anti-cholera toxin monoclonal antibodies were used as the primary antibody and rabbit anti- mouse IgG peroxidase conjugates were used as the secondary antibody. The results revealed that the denatured CT-B expressed in plant cells had protein bands similar to CT-B derived from Vibrio cholerae.

17. Results Banana callus expressing CT-B showed the presence of a protein that migrated to the same position in denaturing gel as the CT-B derived from V. cholerae and was recognized by mouse anti CT-B antibody.

18. Conclusions The young plantlets were separated from the growth medium and transplanted in the garden soil, then allowed to grow under a green house. Banana was chosen because it is a well accepted fruit and could be eaten as a raw fruit. Also, it can be grown in all parts of the world. Edible vaccines were determined to be a very economic and less cost consuming therapy against cholera.