year 12, Issue 4 (September - October 2018)
Iran J Med Microbiol 2018, 12(4): 260-268 |
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1- MSc in Molecular Biotechnology, Department of Genetic Engineering, Research Centre for Sciences and Biotechnology, Malek-Ashtar Industrial University, Tehran, Iran
2- PhD in Molecular Genetics, Assistant Professor, Department of Genetic Engineering, Research Centre for Sciences and Biotechnology, Malek-Ashtar Industrial University, Tehran, Iran , aad.phd.gene@gmail.com
2- PhD in Molecular Genetics, Assistant Professor, Department of Genetic Engineering, Research Centre for Sciences and Biotechnology, Malek-Ashtar Industrial University, Tehran, Iran , aad.phd.gene@gmail.com
Abstract: (4987 Views)
Background and Aims: The virulence plasmid of the Gram negative pathogen Shigella generates many virulence factors within the Ipa-mxi-spa region. Shigella spreads via fecal-oral and person-to-person transmission which causes human bloody diarrhea. In Asia alone, it is estimated that there are 125 million infections and 14,000 deaths due to shigellosis annually. IpaB is essential for Shigella infection and pathogenicity.
Materials and Methods: Expression plasmid based on the araBAD promoter is designed for tight control of background expression and l-arabinose dependent graded expression of the target proteins. IpgC and IpaB genes were amplified using polymerase chain reaction (PCR) with designed primers. In the next step, the products were then cloned into pBAD/myc-HisB expression vector. The presence of the insert was confirmed by restriction digestion. In order to confirm the chaperone role of IpgC on IpaB protein stability, the 300-bp is removed from the N-terminal portion of IpaB and His6-tagged CPD is fused to the C-terminus of target proteins in the pET28b.
Results: The construction of IpaB gene expression plasmid and expression of IpaB protein was achieved. Also, expression of the gene truncation of IpaB along with the His6 tag sequence in the absence of the IpgC gene in Escherichia coli revealed that the role of the IpgC chaperone gene on IpaB expression can be considered as an appropriate strategy for expression of IpaB.
Conclusions: In the present study IpgC and IpaB genes were successfully cloned and expressed under the control of arabinose-dependent promoters to provide IpaB expressing plasmid. The role of the IpgC as a chaperone protein on IpaB expression and stability can be considered as an appropriate strategy for expressing IpaB. The induced vector can be used for future analysis of Shigella vaccine development.
Materials and Methods: Expression plasmid based on the araBAD promoter is designed for tight control of background expression and l-arabinose dependent graded expression of the target proteins. IpgC and IpaB genes were amplified using polymerase chain reaction (PCR) with designed primers. In the next step, the products were then cloned into pBAD/myc-HisB expression vector. The presence of the insert was confirmed by restriction digestion. In order to confirm the chaperone role of IpgC on IpaB protein stability, the 300-bp is removed from the N-terminal portion of IpaB and His6-tagged CPD is fused to the C-terminus of target proteins in the pET28b.
Results: The construction of IpaB gene expression plasmid and expression of IpaB protein was achieved. Also, expression of the gene truncation of IpaB along with the His6 tag sequence in the absence of the IpgC gene in Escherichia coli revealed that the role of the IpgC chaperone gene on IpaB expression can be considered as an appropriate strategy for expression of IpaB.
Conclusions: In the present study IpgC and IpaB genes were successfully cloned and expressed under the control of arabinose-dependent promoters to provide IpaB expressing plasmid. The role of the IpgC as a chaperone protein on IpaB expression and stability can be considered as an appropriate strategy for expressing IpaB. The induced vector can be used for future analysis of Shigella vaccine development.
Type of Study: Original Research Article |
Subject:
Molecular Microbiology
Received: 2018/05/5 | Accepted: 2018/10/22 | ePublished: 2019/01/9
Received: 2018/05/5 | Accepted: 2018/10/22 | ePublished: 2019/01/9
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