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Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli.


ABSTRACT: BACKGROUND:Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain. OBJECTIVES:In the present study, a new truncated form of PD was designed based on conserved areas, and recombinant truncated PD was expressed. MATERIALS AND METHODS:Truncated PD was designed using bioinformatics tools, and a 345 bp fragment of the truncated hpd gene was amplified by polymerase chain reaction (PCR) from H. influenzae and subsequently cloned into the prokaryotic expression vector pBAD-gIIIA. In addition, for the expression of the recombinant protein, the pBAD-truncated PD plasmid was transformed into competent TOP10 cells. The recombinant protein was expressed with Arabinose. The expressed protein was purified by affinity chromatography using Ni-NTA resin. RESULTS:The cloning of PD was confirmed by colony-PCR and enzymatic digestion. Arabinose 0.2% was able to efficiently induce protein expression. The SDS-PAGE analysis showed that our constructed pBAD-PD-TOP10 efficiently produced a target recombinant protein with a molecular weight of 16 kDa. A high concentration of the recombinant protein was obtained via the purification process by affinity chromatography. The recombinant PD was reacted with peroxidase-conjugated rabbit anti-mouse immunoglobulins. CONCLUSIONS:Our results showed that the recombinant protein produced by the pBAD vector in the Escherichia coli system was very efficient.

SUBMITTER: Behrouzi A 

PROVIDER: S-EPMC4600343 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli.

Behrouzi Ava A   Bouzari Saeid S   Siadat Seyed Davar SD   Jafari Anis A   Irani Shiva S  

Jundishapur journal of microbiology 20150829 8


<h4>Background</h4>Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain.<h4>Objectives</h4>In the present study, a new truncated form of PD was designed based on conserved areas, and  ...[more]

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