Faezi, Sobhan, Bahrmand, Ahmad Reza, Siadat, Seyed Davar, Nikokar, Iraj, Sardari, Soroush, Mahdavi, Mehdi. (1396). Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa. , 20(5), 458-466. doi: 10.22038/ijbms.2017.8667
Sobhan Faezi; Ahmad Reza Bahrmand; Seyed Davar Siadat; Iraj Nikokar; Soroush Sardari; Mehdi Mahdavi. "Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa". , 20, 5, 1396, 458-466. doi: 10.22038/ijbms.2017.8667
Faezi, Sobhan, Bahrmand, Ahmad Reza, Siadat, Seyed Davar, Nikokar, Iraj, Sardari, Soroush, Mahdavi, Mehdi. (1396). 'Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa', , 20(5), pp. 458-466. doi: 10.22038/ijbms.2017.8667
Faezi, Sobhan, Bahrmand, Ahmad Reza, Siadat, Seyed Davar, Nikokar, Iraj, Sardari, Soroush, Mahdavi, Mehdi. Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa. , 1396; 20(5): 458-466. doi: 10.22038/ijbms.2017.8667

Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa

Iranian Journal of Basic Medical Sciences
مقاله 2، دوره 20، شماره 5، 2017، صفحه 458-466    اصل مقاله (1.07 M)
نوع مقاله: Original Article
شناسه دیجیتال (DOI): 10.22038/ijbms.2017.8667
نویسندگان
Sobhan Faezi1، 2؛ Ahmad Reza Bahrmand1، 2؛ Seyed Davar Siadat1، 2؛ Iraj Nikokar3؛ Soroush Sardari4؛ Mehdi Mahdavi* 5
1Departments of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran
2Microbiology Research Center (MRC), Pasteur Institute of Iran, Tehran, Iran
3Department of Biotechnology, Laboratory of Microbiology and Immunology of Infectious Diseases, Para Medicine Faculty, Guilan University of Medical Sciences, Rasht, Iran
4Biotechnology Research Center, Drug Design and Bioinformatics Group, Pasteur Institute of Iran, Tehran, Iran
5Departments of Immunology, Pasteur Institute of Iran, Tehran, Iran
چکیده
Objective(s): Infection with Pseudomonas aeruginosa has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel Pseudomonas aeruginosa type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides.
Materials and Methods: We surveyed the expression of the PilQ380-705-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each pilA domains. The  transformants were  expressed in Escherichia coli BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays.
Results: The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results.
Conclusion: Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ380-705-PilA fusion protein could be a reliable candidate vaccine against P. aeruginosa infection.
کلیدواژه‌ها
Fusion protein؛ pET26b؛ PilA؛ PilQ؛ Pseudomonas aeruginosa؛ Type IV pili
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