Expression of Cholera toxin B subunit in Banana callus cultureDawn Rivard
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
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
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
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
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.
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.
Cloning vectorE. coli DH5α PRK2013 together with
pBluescript II KS were used for the initial cloning, sequencing and maintenance of different DNA fragments.
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.
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.
Expression vector
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.
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.
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.
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.
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.
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.
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.