molecular cloning, expression and purification of truncated hpd fragment of haemophilus influenzae in escherichia coli

conclusions our results showed that the recombinant protein produced by the pbad vector in the escherichia coli system was very efficient. 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.