Dataset Information

Design, Expression and Purification of Strongyloides stercoralis IgG4 Immunoreactive Protein (NIE) in Escherichia coli.

ABSTRACT: Background:Strongyloidiasis is a public health concern in northern regions of Iran, caused by Strongyloides stercoralis. Auto-infection cycle can be resulted in high parasitic load, especially in immunocompromised hosts. Because of low sensitivity of stool culture and stool-based microscopy techniques, detection of antibodies in patient's sera can be an alternative diagnostic technique for detection of the nematode. In the present study, as the first step of the development of an ELISA kit for the detection of antibodies against the nematode, IgG4 immunoreactive protein (NIE) was expressed in Escherichia coli expression system, purified and verified. Methods:The NIE gene sequence was retrieved from the GenBank. This sequence was codon-optimized for the expression in E. coli BL21 (DE3). The sequence was inserted into the expression vector pET-30b (+). The recombinant vector was then transferred into competent E. coli BL21 (DE3). Transformed colonies were selected and verified by colony PCR. NIE gene expression was induced with IPTG induction. The protein production was evaluated by SDS-PAGE and verified using Western blotting. Results:The codon-optimized NIE gene had required parameters for expression in E. coli. NIE protein was proved and verified by SDS-PAGE and Western blotting. Conclusion:NIE recombinant protein was successfully expressed in E. coli expression system in appropriate amounts. The recombinant protein can be used for developing ELISA kit in diagnosis of S. stercoralis.

SUBMITTER: Dastan K

PROVIDER: S-EPMC7548455 | biostudies-literature | 2020 Jul-Sep

REPOSITORIES: biostudies-literature

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Design, Expression and Purification of <i>Strongyloides stercoralis</i> IgG4 Immunoreactive Protein (NIE) in <i>Escherichia coli</i>.

Dastan Katayoun K Assmar Mehdi M Amirmozafari Nour N Ghanaei Fariborz Mansour FM Mirpour Mirsasan M

Iranian journal of parasitology 20200701 3

<h4>Background</h4>Strongyloidiasis is a public health concern in northern regions of Iran, caused by <i>Strongyloides stercoralis</i>. Auto-infection cycle can be resulted in high parasitic load, especially in immunocompromised hosts. Because of low sensitivity of stool culture and stool-based microscopy techniques, detection of antibodies in patient's sera can be an alternative diagnostic technique for detection of the nematode. In the present study, as the first step of the development of an ...[more]

PMID: 33082798

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Project description:BackgroundThe diagnosis of strongyloidiasis is challenging. Serological tests are acknowledged to have high sensitivity, but issues due to cross-reactions with other parasites, native parasite antigen supply and intrinsic test variability do occur. Assays based on recombinant antigens could represent an improvement. The aim of this study was to assess the sensitivity and specificity of two novel immunoglobulin (Ig)G and IgG4 enzyme-linked immunosorbent assays (ELISAs) based on the recombinant antigens NIE/SsIR for the diagnosis of strongyloidiasis.MethodsThis was a retrospective diagnostic accuracy study. We included serum samples collected from immigrants from strongyloidiasis endemic areas for whom there was a matched result for Strongyloides stercoralis on agar plate culture and/or PCR assay, or a positive microscopy for S. stercoralis larvae. For the included samples, results were also available from an in-house indirect fluorescent antibody test (IFAT) and a commercial (Bordier ELISA; Bordier Affinity Products SA) ELISA. We excluded: (i) samples with insufficient serum volume; (ii) samples from patients treated with ivermectin in the previous 6 months; and (iii) sera from patients for whom only routine coproparasitology was performed after formol-ether concentration, if negative for S. stercoralis larvae. The performance of the novel assays was assessed against: (i) a primary reference standard, with samples classified as negative/positive on the basis of the results of fecal tests; (ii) a composite reference standard (CRS), which also considered patients to be positive who had concordant positive results for the IFAT and Bordier ELISA or with a single "high titer" positive result for the IFAT or Bordier ELISA. Samples with a single positive test, either for the IFAT or Bordier ELISA, at low titer, were considered to be "indeterminate," and analyses were carried out with and without their inclusion.ResultsWhen assessed against the primary reference standard, the sensitivities of the IgG and IgG4 ELISAs were 92% (95% confidence interval [CI]: 88-97%) and 81% (95% CI: 74-87%), respectively, and the specificities were 91% (95% CI: 88-95%) and 94% (95% CI: 91-97%), respectively. When tested against the CRS, the IgG ELISA performed best, with 78% sensitivity (95% CI: 72-83%) and 98% specificity (95% CI: 96-100%), when a cut-off of 0.675 was applied and the indeterminate samples were excluded from the analysis.ConclusionThe NIE-SsIR IgG ELISA demonstrated better accuracy than the IgG4 assay and was deemed promising particularly for serosurveys in endemic areas.

Project description:E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2-4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions.

Dataset Information

Design, Expression and Purification of Strongyloides stercoralis IgG4 Immunoreactive Protein (NIE) in Escherichia coli.

Publications

Design, Expression and Purification of <i>Strongyloides stercoralis</i> IgG4 Immunoreactive Protein (NIE) in <i>Escherichia coli</i>.

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