Cloning, Expression, and Purification of Pseudomonas aeruginosa Flagellin, and Characterization of the Elicited Anti-Flagellin Antibody
نویسندگان
1 Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, IR Iran
2 Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, IR Iran
3 Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran
4 Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran
5 1Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran
6
doi
چکیده
Pseudomonas aeruginosa is an important opportunistic human pathogen that causes serious infections in immunocompromisedhosts. The single polar flagellum is an important factor in both virulence and colonization.Objectives: 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 ofthe rabbit polyclonal antibody raised against this r-B-flagellin.Materials and Methods: In the current experimental study, the r-B-flagellin gene was isolated from the P. aeruginosa PAO1 strain byPCR. It was cloned into the pET-28a vector and then transformed into the E. coli BL21 strain. Next, r-B-flagellin was overexpressed andaffinity purified by Ni-NTA agarose-affinity chromatography, followed by on-column resolubilization. Polyclonal antisera againstthe recombinant flagellin were raised in rabbits, and the functional activity of the anti-r-B-flagellin antibody was determined by invitro 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 opsonophagocytosisof 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 againstit exhibited functional activity.
