Avicenna J Med Biotech

arij002

Avicenna Journal of Medical Biotechnology

2008-2835 2008-4625

Avicenna Research Institute

ajmb302

Molecular Characterization and Functional Analysis of the PilQ380-705: a Novel Secretin Domain in Pseudomonas aeruginosa

Faezi

Sobhan

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Nikokar

Iraj

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Elmi

Ali

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Ghasemi

Yusuf

Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

Farahbakhsh

Mojtaba

Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan, IranDepartment of Medicinal Chemistry, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran

Salimi Chirani

Alireza

Mahdavi

Mehdi

Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, IranKhatam University, Tehran, Iran

10 1 34 40 14 9 2016 15 2 2017

Background: Type 4 pili (T4P) is an important virulence factor of Pseudomonas aeruginosa (P. aeruginosa). T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ (PilQ380-706) was produced as a recombinant protein. Methods: A 981 bp fragment of C-terminal of the pilQ secretin (pilQ1138-2118) from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ1138-2118 gene in the recombinant construct (pET28a/pilQ) was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ380-706 confirmed by Western blot analysis and twitching inhibition assay.  Results: The PCR and enzymatic digestion results showed the presence of the pilQ1138-2118 gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ380-706 is approximately 37 kDa. The Western blot analysis confirmed the specificity of specific IgG against the PilQ380-706 protein. The PilQ380-706 protein showed high biological activity in all of these standard assays. Conclusion: Since, the PilQ380-706 protein plays an important role in the biogenesis of pili; and thus, the primary establishment of P. aeruginosa; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies.