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Molecular Characterization and Functional Analysis of the PilQ380-705: a Novel Secretin Domain in Pseudomonas aeruginosa
<p>Background: Type 4 pili (T4P) is an important virulence factor of<em> Pseudomonas aeruginosa (P. aeruginosa)</em>. 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 (PilQ<sub>380-706</sub>) was produced as a recombinant protein.<br />
Methods: A 981 <em>bp</em> fragment of C-terminal of the <em>pilQ</em> secretin (<em>pilQ</em><sub>1138-2118</sub>) from was designed into the prokaryotic expression vector pET28a. The presence of the <em>pilQ</em><sub>1138-2118</sub> gene in the recombinant construct (pET28a/<em>pilQ</em>) was assessed by double digestion and PCR. After transformation, expression of the recombinant <em>PilQ</em> 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 <em>PilQ</em><sub>380-706</sub> confirmed by Western blot analysis and twitching inhibition assay. <br />
Results: The PCR and enzymatic digestion results showed the presence of the <em>pilQ</em><sub>1138-2118</sub> gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant <em>PilQ</em><sub>380-706</sub> is approximately 37 <em>kDa</em>. The Western blot analysis confirmed the specificity of specific IgG against the <em>PilQ</em><sub>380-706</sub> protein. The<em> PilQ</em><sub>380-706</sub> protein showed high biological activity in all of these standard assays.<br />
Conclusion: Since, the <em>PilQ</em><sub>380-706</sub> protein plays an important role in the biogenesis of pili; and thus, the primary establishment of <em>P. aeruginosa</em>; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies.</p>
Pseudomonas aeruginosa, Proteins, Secretin
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https://www.ajmb.org/En/Article.aspx?id=302
https://www.ajmb.org/PDF/En/FullText/302.pdf
SobhanFaeziMycobacteriology Research Center (MRC), Tehran, Iran1203
IrajNikokarLaboratory of Microbiology and Immunology of Infectious Diseases, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1204
AliElmiMedical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1205
YusufGhasemiMedical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1206
MojtabaFarahbakhshMedical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1207
AlirezaSalimi ChiraniDepartment of Medical Microbiology, Faculty of Medicine, Shahid Beheshty University of Medical Science, Tehran, Iran11337
MehdiMahdaviDepartment of Immunology, Pasteur Institute of Iran, Tehran, Iran594