Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation

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, we want to produce LTB protein which encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as a antigen in next studies. 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 transgenic centella plant. 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 transformated into centella plants by 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.

Construction of the recombinant Escherichia coli strain producing a homologous thermolabile toxin subunit

“…Введение. Основными факторами вирулентности энтеропатогенных штаммов Escherichia coli являются факторы адгезии, способствующие колонизации бактериями энтероцитов тонкого отдела кишечника, а также способность к продукции термолабильного [1][2][3] и термостабильного токсинов. Термолабильные (LT) и/или термостабильные токсины вызывают секрецию энтероцитами жидкости и электролитов, что сопровождается водянистой диареей, истощением и гибелью человека или животного.…”