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

en 29296265 Molecular Characterization and Functional Analysis of the PilQ380-705: a Novel Secretin Domain in Pseudomonas aeruginosa 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. Pseudomonas aeruginosa, Proteins, Secretin 34 40 https://www.ajmb.org/En/Article.aspx?id=302 https://www.ajmb.org/PDF/En/FullText/302.pdf SobhanFaeziMycobacteriology Research Center (MRC), Tehran, Iran1203IrajNikokarLaboratory of Microbiology and Immunology of Infectious Diseases, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1204AliElmiMedical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1205YusufGhasemiMedical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1206MojtabaFarahbakhshMedical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran1207AlirezaSalimi ChiraniDepartment of Medical Microbiology, Faculty of Medicine, Shahid Beheshty University of Medical Science, Tehran, Iran11337MehdiMahdaviDepartment of Immunology, Pasteur Institute of Iran, Tehran, Iran594