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Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile B subunit of enterotoxigenic Escherichia coli.

Alerasol M, Mousavi Gargari SL, Nazarian S, Bagheri S - Iran. Biomed. J. (2014)

Bottom Line: Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells.The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response.The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Shahed University, Tehran, Iran. slmousavi@shahed.ac.ir.

ABSTRACT

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate.

Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated.

Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM1 binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells.

Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation.

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Related in: MedlinePlus

Purification of recombinant proteins with nickel-nitrilotriacetic acid column. Lane 1, expressed protein before purification; lane 2, protein molecular marker; lane 3, flow-through; lanes 4 and 5, wash column with C and D buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl); lane 6, purified protein after elution with Elution buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl), and lane 7, wash column MES (2-N-morpholine-ethanesulfonic acid) buffer
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Figure 3: Purification of recombinant proteins with nickel-nitrilotriacetic acid column. Lane 1, expressed protein before purification; lane 2, protein molecular marker; lane 3, flow-through; lanes 4 and 5, wash column with C and D buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl); lane 6, purified protein after elution with Elution buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl), and lane 7, wash column MES (2-N-morpholine-ethanesulfonic acid) buffer

Mentions: Expression and purification of recombinant protein. The synthetic chimeric gene was expressed in E. coli (BL21DE3) with the N-terminal 6×-His tag and analyzed by SDS-PAGE (Fig. 2). The SDS-PAGE analysis showed the presence of a 33-KD recombinant chimeric protein. Purification of the recombinant chimeric protein was carried out under denaturing conditions, and SDS-PAGE analysis revealed the presence of the protein as a major band (Fig. 3). The expression of recombinant chimeric protein was confirmed by Western blotting using anti-His tag antibodies.


Immunogenicity of a fusion protein comprising coli surface antigen 3 and labile B subunit of enterotoxigenic Escherichia coli.

Alerasol M, Mousavi Gargari SL, Nazarian S, Bagheri S - Iran. Biomed. J. (2014)

Purification of recombinant proteins with nickel-nitrilotriacetic acid column. Lane 1, expressed protein before purification; lane 2, protein molecular marker; lane 3, flow-through; lanes 4 and 5, wash column with C and D buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl); lane 6, purified protein after elution with Elution buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl), and lane 7, wash column MES (2-N-morpholine-ethanesulfonic acid) buffer
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225060&req=5

Figure 3: Purification of recombinant proteins with nickel-nitrilotriacetic acid column. Lane 1, expressed protein before purification; lane 2, protein molecular marker; lane 3, flow-through; lanes 4 and 5, wash column with C and D buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl); lane 6, purified protein after elution with Elution buffer (8M Urea, 20 mM NaH2PO4, 500 mM NaCl), and lane 7, wash column MES (2-N-morpholine-ethanesulfonic acid) buffer
Mentions: Expression and purification of recombinant protein. The synthetic chimeric gene was expressed in E. coli (BL21DE3) with the N-terminal 6×-His tag and analyzed by SDS-PAGE (Fig. 2). The SDS-PAGE analysis showed the presence of a 33-KD recombinant chimeric protein. Purification of the recombinant chimeric protein was carried out under denaturing conditions, and SDS-PAGE analysis revealed the presence of the protein as a major band (Fig. 3). The expression of recombinant chimeric protein was confirmed by Western blotting using anti-His tag antibodies.

Bottom Line: Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells.The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response.The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Shahed University, Tehran, Iran. slmousavi@shahed.ac.ir.

ABSTRACT

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate.

Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated.

Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM1 binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells.

Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation.

Show MeSH
Related in: MedlinePlus