Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody.
BACKGROUND
Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromised hosts. The single polar flagellum is an important factor in both virulence and colonization.


OBJECTIVE
As flagellin is the major component of the flagellar ﬁlament, the main aims of the present study are to identify, clone, express, and purify the recombinant type B flagellin (r-B-flagellin) of P. aeruginosa, as well as to evaluate the functional activity of the rabbit polyclonal antibody raised against this r-B-flagellin.


METHODS
In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain by PCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed and affinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera against the recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by in vitro assays.


RESULTS
The polyclonal antibodies raised against this r-B-flagellin inhibited the motility of the homologous PAO1 strain of P. aeruginosa, which significantly decreased the invasion of the PAO1 strain into the A549 cells and also enhanced the opsonophagocytosis of this strain. However, our polyclonal antibody showed little effect on the heterologous PAK strain.


CONCLUSIONS
The r-B-flagellin carried antigenic epitopes just like the native flagellin, while the polyclonal antibody raised against it exhibited functional activity.
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran.
Bahador
B
Behrouz
Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, IR Iran.
Nour
N
Amirmozafari
Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
Nima
N
Khoramabadi
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran.
Mahboobeh
M
Bahroudi
Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, IR Iran.
Parisa
P
Legaee
Department of Immunology, Pasteur Institute of Iran, Tehran, IR Iran.
Mehdi
M
Mahdavi
Polyclonal Antibody; Pseudomonas aeruginosa; Recombinant Flagellin