Molecular characterization and Functional Analysis of the PilQ 380-706 : a Novel Secretin Domain in Pseudomonas aeruginosa .

Autor: Faezi S; Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran.; Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran.; Mycobacteriology Research Center (MRC), Tehran, Iran., Nikokar I; Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran.; Laboratory of Microbiology and Immunology of Infectious Diseases, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran., Elmi A; Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran., Ghasemi Y; Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran., Farahbakhsh M; Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences, Rasht, Iran., Salimi Chirani A; Department of Medical Microbiology, Faculty of Medicine, Shahid Beheshty University of Medical Science, Tehran, Iran., Mahdavi M; Department of Immunology, Pasteur Institute of Iran, Tehran, Iran.
Jazyk: angličtina
Zdroj: Avicenna journal of medical biotechnology [Avicenna J Med Biotechnol] 2018 Jan-Mar; Vol. 10 (1), pp. 34-40.
Abstrakt: 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 (PilQ 380-706 ) was produced as a recombinant protein.
Methods: A 981 bp fragment of C-terminal of the pilQ secretin ( pilQ 1138-2118 ) from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ 1138-2118 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 PilQ 380-706 confirmed by Western blot analysis and twitching inhibition assay.
Results: The PCR and enzymatic digestion results showed the presence of the pilQ 1138-2118 gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ 380-706 is approximately 37 kDa . The Western blot analysis confirmed the specificity of specific IgG against the PilQ 380-706 protein. The PilQ 380-706 protein showed high biological activity in all of these standard assays.
Conclusion: Since, the PilQ 380-706 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.
Databáze: MEDLINE
Externí odkaz: