Alessandra Cavalcante

Luís Morais Mariúba

Maria dos Santos

Paulo Nogueira

BMC Proceedings

PubMed

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Expression of proteins Heat-Labile (Lt-1) and Heat-Stable (STa) Escherichia coli expression of proteins Heat-Labile (Lt-1) and Heat-Stable (STa) Escherichia coli

POSTER PRESENTATION Open Access
Expression of proteins Heat-Labile (Lt-1) and
Heat-Stable (STa) Escherichia coli expression of
proteins Heat-Labile (Lt-1) and Heat-Stable (STa)
Escherichia coli
Alessandra Cavalcante*, Maria Eliza Caldas dos Santos, Luís André Morais Mariúba, Paulo Afonso Nogueira
From 5th Congress of the Brazilian Biotechnology Society (SBBIOTEC)
Florianópolis, Brazil. 10-14 November 2013
Introduction
Escherichia coli has a direct relationship with the diar-
rheagenic diseases, which occur quite in developing
countries due to poor sanitation system and public
health. It mainly affects children, causing a rate of mor-
bidity and mortality worrying.
This bacterium has a versatility of strains expressing
different mechanisms and virulence factors, thus the
importance in studying the taxonomy of these cepas.
Classified into six categories: enteropathogenic E.coli
(EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E.
coli (EIEC), enterohemorrhagic E. coli (EHEC), enteroag-
gregative E. coli (EAEC) and diffusely adhering E. coli
(DAEC). Among them, ETEC is the most common etio-
logic agent of infantile diarrhea related and has the
characteristic resist peristaltic movements and adhere to
the small intestine produce enterotoxin. The enterotox-
ins are proteins encoded by plasmids and occurring in
two forms: one stable toxin (ST) which may be of the
type Sta and Stb or and a labile toxin (LT), which also
can be of two types LT and LT-1 or LT -2. Strains of
ETEC causes diarrhea when adhere to enterocytes of the
small intestine, mediated by one or more adhesins called
CFA (colonization factor antigens).
The development of antisera and specific antibodies
identification of species and subspecies of Enterobacter-
iaceae prevent the misuse of antibiotics, will enable the
taxonomic study and facilitate medical diagnosis.
Methods
This study samples of enterotoxigenic E. coli (ETEC) are
used the bank’s sample Institute Leônidas and Maria
Deane (ILMD-FIOCRUZ). After extraction of genomic
DNA samples, was made the standardization of PCR.
The result was visualized on 1.5% agarose gel and bands
for the purification of LT-1 and Sta genes was used
QIAquick Gel Extraction Kit QIAGEN. Then, the pre-
paration of competent cell was made with top 10 for
cloning and subcloning, DHa5 pLysS and BL21 for
expression. Purified fragments were ligated to the vector
pGEM T Easy cloning and transformation was per-
formed. The white colonies obtained after transforma-
tion of bacteria with binding were subjected to
screening by PCR and sent for sequencing. Plasmids
were digested by the restriction enzymes EcoR1 and
BamH1 cloning vector and pQE (Invitrogen) was
digested using enzymes Sph1 and Pst1. Fragments
linked to expression vectors were inserted by transfor-
mation into BL21 pLysS plasmid and subjected to
extraction. Confirmation of the expression of target pro-
teins was performed by SDS-PAGE and confirmation of
gene expression was used Western blot (Invitrogen).
Results
Confirmation of cloning was performed by PCR with
sense and antisense primer pGEM of the genes chosen
allowing increasing the size of the fragment. In the sub-
cloning, we used the restriction enzyme EcoR1 digested
that the correct gene Sta, however digested LT-1 gene
sequences not expected. Due to this gene for the expres-
sion of LT will use an expression plasmid (pQE) different.
Instituto Leônidas e Maria Deane, Manaus, Amazonas, Brasil
Cavalcante et al. BMC Proceedings 2014, 8(Suppl 4):P43
http://www.biomedcentral.com/1753-6561/8/S4/P43
© 2014 Cavalcante et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Protein expression heat-stable Sta using plasmid
pRSETC was seen in Western Blot presenting highest
expression pellets and size of 4 kDA.
Published: 1 October 2014
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doi:10.1186/1753-6561-8-S4-P43
Cite this article as: Cavalcante et al.: Expression of proteins Heat-Labile
(Lt-1) and Heat-Stable (STa) Escherichia coli expression of proteins Heat-
Labile (Lt-1) and Heat-Stable (STa) Escherichia coli. BMC Proceedings 2014
8(Suppl 4):P43.
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Cavalcante et al. BMC Proceedings 2014, 8(Suppl 4):P43
http://www.biomedcentral.com/1753-6561/8/S4/P43
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Expression of proteins Heat-Labile (Lt-1) and Heat-Stable (STa) Escherichia coli expression of proteins Heat-Labile (Lt-1) and Heat-Stable (STa) Escherichia coli

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