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Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants
Author(s) -
RosalesMendoza Sergio,
AlpucheSolís Ángel Gabriel,
SoriaGuerra Ruth Elena,
MorenoFierros Leticia,
MartínezGonzález Luzmila,
HerreraDíaz Areli,
Korban Schuyler S.
Publication year - 2009
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2008.03666.x
Subject(s) - biology , enterotoxigenic escherichia coli , cholera toxin , western blot , fusion protein , toxin , antigenicity , microbiology and biotechnology , protein subunit , escherichia coli , virology , antigen , enterotoxin , gene , recombinant dna , biochemistry , genetics
Summary Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat‐stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB‐ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant‐based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB‐ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1‐ELISA revealed that the LTB‐ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17‐KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze‐dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB‐ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB‐ST‐containing tobacco. These findings suggest that tobacco plants expressing LTB‐ST could serve as a plant‐based candidate vaccine model providing broad‐spectrum protection against ETEC‐induced diarrhoeal disease.