Production of recombinant 14-3-3 protein and determination of its immunogenicity for application in serodiagnosis of strongyloidiasis.
14-3-3 protein
immunoreactivity, recombinant protein
serodiagnosis, Strongyloides stercoralis
Journal
Transactions of the Royal Society of Tropical Medicine and Hygiene
ISSN: 1878-3503
Titre abrégé: Trans R Soc Trop Med Hyg
Pays: England
ID NLM: 7506129
Informations de publication
Date de publication:
01 06 2019
01 06 2019
Historique:
received:
04
09
2018
revised:
16
11
2018
accepted:
03
02
2019
pubmed:
12
3
2019
medline:
27
6
2020
entrez:
12
3
2019
Statut:
ppublish
Résumé
Strongyloides stercoralis is the fourth most important intestinal nematode worldwide. The parasite load and larvae count are often low, thus conventional methods are not sufficiently sensitive to detect the infection. In this study we developed an immunoglobulin G-based enzyme-linked immunosorbent assay (ELISA) method to detect antibodies against S. stercoralis 14-3-3 protein in patients' sera. S. stercoralis RNA was extracted and following complementary DNA synthesis, the 708-bp fragment of 14-3-3 protein was amplified by polymerase chain reaction and cloned into the pET28a+ expression vector. The 30-kDa recombinant 14-3-3 protein was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Finally, its immunoreactivity was assessed by indirect ELISA and western blotting. The S. stercoralis 14-3-3 gene was successfully amplified and cloned into an expression vector. The 30-kDa recombinant protein was purified by affinity chromatography. An ELISA developed in-house detected infected patients' sera with 96% sensitivity. We concluded that the recombinant 14-3-3 protein has enough sensitivity and specificity for detection of strongyloidiasis in human sera and could be applied for serodiagnosis.
Sections du résumé
BACKGROUND
Strongyloides stercoralis is the fourth most important intestinal nematode worldwide. The parasite load and larvae count are often low, thus conventional methods are not sufficiently sensitive to detect the infection. In this study we developed an immunoglobulin G-based enzyme-linked immunosorbent assay (ELISA) method to detect antibodies against S. stercoralis 14-3-3 protein in patients' sera.
METHODS
S. stercoralis RNA was extracted and following complementary DNA synthesis, the 708-bp fragment of 14-3-3 protein was amplified by polymerase chain reaction and cloned into the pET28a+ expression vector. The 30-kDa recombinant 14-3-3 protein was expressed in Escherichia coli BL21 (DE3) cells and purified by affinity chromatography. Finally, its immunoreactivity was assessed by indirect ELISA and western blotting.
RESULTS
The S. stercoralis 14-3-3 gene was successfully amplified and cloned into an expression vector. The 30-kDa recombinant protein was purified by affinity chromatography. An ELISA developed in-house detected infected patients' sera with 96% sensitivity.
CONCLUSIONS
We concluded that the recombinant 14-3-3 protein has enough sensitivity and specificity for detection of strongyloidiasis in human sera and could be applied for serodiagnosis.
Identifiants
pubmed: 30856271
pii: 5374605
doi: 10.1093/trstmh/trz006
doi:
Substances chimiques
14-3-3 Proteins
0
Immunoglobulin G
0
Recombinant Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
326-331Informations de copyright
© The Author(s) 2019. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.