Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli
| Author | Ava Behrouzi | en |
| Author | Saeid Bouzari | en |
| Author | Seyed Davar Siadat | en |
| Author | Anis Jafari | en |
| Author | Shiva Irani | en |
| Orcid | Seyed Davar Siadat [0000-0002-6892-5603] | en |
| Issued Date | 2015-08-01 | en |
| 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. | en |
| DOI | https://doi.org/10.5812/jjm.23218 | en |
| Keyword | <i>Haemophilus influenzae</i> | en |
| Keyword | Recombinant Protein | en |
| Keyword | Expression Vector | en |
| Keyword | Ni-NTA | en |
| Publisher | Brieflands | en |
| Title | Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli | en |
| Type | Research Article | en |