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

Expression of Cholera toxin B subunit in Banana callus culture

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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 - PowerPoint PPT Presentation

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Page 1: Expression of Cholera toxin B subunit in Banana callus culture

Expression of Cholera toxin B subunit in Banana callus cultureDawn Rivard

Page 2: Expression of Cholera toxin B subunit in Banana callus culture

CholeraAn infection of the small

intestine caused by the bacterium Vibrio cholerae◦A gram negative comma-shaped

bacterium with a polar flagellumThe 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

Page 3: Expression of Cholera toxin B subunit in Banana callus culture

Cholera toxinAn 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

bondThe B subunits form a

five-membered ring and part A forms an extended alpha helix which fits in the central pore of the ring

Page 4: Expression of Cholera toxin B subunit in Banana callus culture

Mechanism of actionThe 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

Page 5: Expression of Cholera toxin B subunit in Banana callus culture

Mechanism of actionPart 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

Page 6: Expression of Cholera toxin B subunit in Banana callus culture

ExperimentIn 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.

Page 7: Expression of Cholera toxin B subunit in Banana callus culture

ExperimentThe 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.

Page 8: Expression of Cholera toxin B subunit in Banana callus culture

Cloning vectorE. coli DH5α PRK2013 together with

pBluescript II KS were used for the initial cloning, sequencing and maintenance of different DNA fragments.

Page 9: Expression of Cholera toxin B subunit in Banana callus culture

Cloning vectorDNA 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.

Page 10: Expression of Cholera toxin B subunit in Banana callus culture

Expression vectorThe 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.

Page 11: Expression of Cholera toxin B subunit in Banana callus culture

Expression vector

Page 12: Expression of Cholera toxin B subunit in Banana callus culture

ResultsThe 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.

Page 13: Expression of Cholera toxin B subunit in Banana callus culture

ResultsThe 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.

Page 14: Expression of Cholera toxin B subunit in Banana callus culture

ResultsIntegration 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.

Page 15: Expression of Cholera toxin B subunit in Banana callus culture

ResultsThe 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.

Page 16: Expression of Cholera toxin B subunit in Banana callus culture

ResultsAnti-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.

Page 17: Expression of Cholera toxin B subunit in Banana callus culture

ResultsBanana 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.

Page 18: Expression of Cholera toxin B subunit in Banana callus culture

ConclusionsThe 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.