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Expression of Cholera toxin B subunit in Banana callus culture

Expression of Cholera toxin B subunit in Banana callus culture. Dawn Rivard. Cholera. An infection of the small intestine caused by the bacterium Vibrio cholerae A gram negative comma-shaped bacterium with a polar flagellum

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Expression of Cholera toxin B subunit in Banana callus culture

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  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 Vibriocholerae • 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 H₂O, 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 Vibriocholerae. • 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 DH5α 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 Agrobacteriumtumefaciens.

  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 IgGperoxidase 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 Vibriocholerae.

  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.

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