Cloning, expression, purification, and characterization of recombinant flagellin isolated from Pseudomonas aeruginosa.
Pseudomonas aeruginosa as an opportunistic pathogen causes lethal infections in immunocompromised individuals. This bacterium possesses a polar flagellum made up of flagellin subunits. Flagella have important roles in motility, chemotaxis, and establishment of P. aeruginosa in acute phase of infections. Isolation, cloning, and expression of flagellin were aimed at in this study. Flagellin gene (fliC) of P. aeruginosa strain 8821M was isolated by PCR and cloned into a pET expression vector. The recombinant flagellin (46 kDa) was overexpressed as inclusion bodies (IBs). IBs were solubilized in guanidine hydrochloride (GuHCl) followed by affinity-purification and renatured using Ni(2+)-Sepharose resin. Recombinant flagellins reacted with the serum from a rabbit previously immunized with native flagellin. In addition, polyclonal antiserum raised against the recombinant flagellin was shown to significantly inhibit the cell motility of P. aeruginosa strain 8821M in vitro.
Goudarzi
Gholamreza
G
Department of Bacteriology, Medical Sciences School, Tarbiat Modares University, Tehran, Iran.
Sattari
Morteza
M
Roudkenar
Mehryar Habibi
MH
Montajabi-Niyat
Mehran
M
Zavaran-Hosseini
Ahmad
A
Mosavi-Hosseini
Kamran
K
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D005408:Flagellin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D007793:Lagomorpha; D008124:Locomotion / Q000276:immunology; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000276:immunology
D000907:Antibodies, Bacterial; D005408:Flagellin