Title:Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation
Volume: 21
Issue: 10
Author(s): Nguyen H. Loc*, Nghiem V. Tung, Phan T.A. Kim and Moon S. Yang
Affiliation:
- Institute of Bioactive Compounds and Department of Biotechnology, University of Sciences, Hue University, Hue, Thua Thien Hue 530000,Vietnam
Keywords:
Biolistic transformation, Centella asiatica, heat-labile enterotoxin B subunit of E. coli (LTB), plant-based vaccine,
synthetic ltb gene (s-ltb).
Abstract:
Background: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a
non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant
which can induce a strong immune response against co-administered antigens.
Objective: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb,
s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed.
Methods: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV
35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification
was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression
of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB
protein was determined by GM1-ELISA.
Results: PCR amplification has found seven transgenic centella individuals. However, only five of
them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected
in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated
that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits
formed active pentamers.
Conclusion: The s-ltb gene that was successfully transformed into centella plants by the biolistic
method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure
that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.
