Bioinformatical Study and Evaluation of Expression of Cholera Toxin Subunit B Optimized Gene as a Vaccine Candidate

Document Type : Original Article (s)

Authors

1 Biology Research Centre, School of Basic Science, Imam Hossein University, Tehran, Iran

2 PhD Student in Nanobiotechnology, Biology Research Centre, School of Basic Science, Imam Hossein University, Tehran, Iran

3 Applied Biotechnology Research Centre, Baqitatolah University of Medical Sciences, Tehran, Iran

Abstract

Background: Cholera is a lethal diarrheal disease caused by Vibrio cholerae. Cholera toxin is the major virulence factor in pathogenesis of Vibrio cholerae. The B subunit of the enterotoxin, which is responsible for toxin binding to eukaryotic cells, has immunogenic properties. The aim of this study was bioinformatic investigation and production of recombinant cholera toxin subunit B (CtxB).Methods: CtxB gene was analysed for rare codons and gene optimization was performed using optimization software. Recombinant pET28a/ctxb plasmid was transformed to E. coli BL21 DE3 and expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). The protein expression was evaluated using Sodium dodecyl sulphate- Polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting analysis. The recombinant protein was purified using Nickel-Nitrilotriacetic acid (Ni–NTA) affinity chromatography.Findings: Codon adaptation index (CAI) on the native gene was 0.61, while the optimized sequence had a CAI of 0.92. Percentage of codon having high-frequency distribution was improved to 67%. Restriction analysis confirmed cloning of the CtxB gene into pET28a vector. Western blotting showed specific reactivity of recombinant protein with anti-CTX antibody. The total yield of purified protein was 9 mg/l.Conclusion: The results indicated that CtxB gene optimization is a useful approach to high-level expression of recombinant protein. The protein could be produced in capsulated form for oral immunization purpose.

Keywords

References

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Journal of Isfahan Medical School
Volume 32, Issue 279 - Serial Number 279
January and February 2014
Pages 378-387

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History

  • Receive Date: 10 February 2014
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

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