Shigella dysentery stxA mutant (E167Q-A231D-G234E) gene design and optimization of recombinant protein expression and purification

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Abstract

Abstract:


Objective: Shigella dysentery by producing shigatoxin (subunits A, B) is one of the most important human pathogenic intestinal bacteria. Entring to epithelial cells, the toxin inhibits protein synthesis leading to cell deat. In spite of great investigation on vaccine production against S.dysentery studying to achieve significant stxA recombinant protein still remains important.The objective of this study was designing mutant stxA gene and expressing protein production as vaccine candidate against stx for further immunization studies.

Methods: three stxA mutant gene including (E167Q-A231D-G234E) were designed and the synthetic gene in pET28a plasmid was obtained and confirmed by PCR. Thereafter the plasmid was transformed into the host cell E.coli BL21 DE3 after which gene expression was optimized and protein purity assay was then performe.
Results: preliminary studies led to mutant stop gene design after which it was confirmed by synthetic plasmid and PCR.The Expression of this gene in BL21 DE3 host cell was them optimized which was resulted in forming large amount of protein inclusion bodies.purification of inclusion bodies and protein solubilization was performed with a combinatorial method.
Conclusion and discussion: regarding the mechanism of shigatoxin effect and simultaneous use of two different mutations in this gene less toxicity is expected in comparison with previous mutants as vaccine candidate, posing a better vaccine candidate.

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History

  • Receive Date: 30 January 2019
  • Revise Date: 25 November 2024
  • Accept Date: 30 January 2019
  • Publish Date: 20 March 2012

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