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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

Modeled structure of chimeric protein by I-TASSER software
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Figure 1: Modeled structure of chimeric protein by I-TASSER software

Mentions: Design and construction of chimeric gene. A synthetic chimeric sequence encoding the cstH and eltB genes was designed using E. coli codon bias. To optimize the synthetic gene, negative cis acting motifs and repeated sequences were avoided. Both the wild type and the synthetic chimera were analyzed for their codon bias and GC content. The overall GC content was improved from 38.96% to 48.75% upon codon optimization, which increased the overall stability of mRNA. ΔG of the best predicted structure was -147.5 kcal/mol. The nucleotides at the starting of the 5′ did not have a long stable hairpin or pseudoknot, whereas in the native mRNA, the ΔG was -112 kcal/mol. The chimeric gene showed a codon adaptation index of 0.96 compared to that of the wild-type gene, which was only 0.72. Ab initio modeling of the synthetic sequence was exploited to produce three dimensional models of the chimeric protein. The result of tertiary structure of the chimeric protein construction using I-TASSER showed a protein with two main domains linked together with a linker (Fig. 1).


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)

Modeled structure of chimeric protein by I-TASSER software
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Modeled structure of chimeric protein by I-TASSER software
Mentions: Design and construction of chimeric gene. A synthetic chimeric sequence encoding the cstH and eltB genes was designed using E. coli codon bias. To optimize the synthetic gene, negative cis acting motifs and repeated sequences were avoided. Both the wild type and the synthetic chimera were analyzed for their codon bias and GC content. The overall GC content was improved from 38.96% to 48.75% upon codon optimization, which increased the overall stability of mRNA. ΔG of the best predicted structure was -147.5 kcal/mol. The nucleotides at the starting of the 5′ did not have a long stable hairpin or pseudoknot, whereas in the native mRNA, the ΔG was -112 kcal/mol. The chimeric gene showed a codon adaptation index of 0.96 compared to that of the wild-type gene, which was only 0.72. Ab initio modeling of the synthetic sequence was exploited to produce three dimensional models of the chimeric protein. The result of tertiary structure of the chimeric protein construction using I-TASSER showed a protein with two main domains linked together with a linker (Fig. 1).

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