摘要
AIM To study immunogenicity of Pseudomonas N terminal flagellin as an adjuvant for Acinetobacter baumannii(A. baumanni) biofilm associated protein(Bap).METHODS The N terminal flagellin gene was amplified. The p ET28a(+) and polymerase chain reaction products weredigested with HindⅢ and Eco R Ⅰ. The ligation of N terminal flagellin into p ET28a(?+) was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21(DE3) as a suitable expression host. p ET28a(?+) vector harboring a conserved region of Bap from our previous work was used. The recombinant proteins were expressed, analyzed by SDS-PAGE method and was purified by affinity chromatography with His-Tag residues followed by confirmation with western blotting. Mice were immunized with recombinant N terminal flagellin and Bap subunits. The immunized animals were intranasally(i.n) challenged with A. baumannii and Pseudomonas aeruginosa(P. aeruginosa).RESULTS The flagellin enhanced the immunogenicity of Bap causing an increase in specific Ig G titers in serum(P < 0.001). Internal organs, i.e., liver, lung and spleen of the BapFlagellin immunized group challenged with A. baumannii showed significantly lower bacterial load compared to the control group. The bacterial loads were studied in internal organs. A. baumannii infected immunized animals with Bap-Flagellin exhibited internal organs with minor bacterial load while P. aeruginosa PAO1 infected group showed heavy bacterial load of(4.3 ± 0.12) × 106,(1.1 ± 0.01) × 106 and(2.2 ± 0.22) × 106 per gram of lungs, liver and spleen respectively. Bacterial loads were detected per gram of lungs, liver and spleen of the mice group immunized with Bap were(1.2 ± 0.06) × 107,(11.1 ± 0.041) × 105 and(3.6 ± 0.42) × 106 respectively. In vivo neutralization assay indicated that all experimental mice groups, except for Flagellin administered group was significantly(P < 0.05) protected against A. baumannii. CONCLUSION These results demonstrate that P. aeruginosa Flagellin as an adjuvant for Bap A. baumannii could be a useful model to evaluate new vaccine against A. baumannii.
AIMTo study immunogenicity of Pseudomonas N terminal fagellin as an adjuvant for Acinetobacter baumannii （A. baumannii ） bioflm associated protein （Bap）.METHODSThe N terminal fagellin gene was amplifed. The pET28a （＋） and polymerase chain reaction products were digested with HindⅢ and EcoR Ⅰ. The ligation of N terminal flagellin into pET28a （＋） was performed using T4 DNA ligase and was then transformed into Escherichia coli BL21 （DE3） as a suitable expression host. pET28a （＋） vector harboring a conserved region of Bap from our previous work was used. The recombinant proteins were expressed, analyzed by SDS-PAGE method and was purifed by affnity chromatography with His-Tag residues followed by confrmation with western blotting. Mice were immunized with recombinant N terminal flagellin and Bap subunits. The immunized animals were intranasally （i.n） challenged with A. baumannii and Pseudomonas aeruginosa （ P. aeruginosa）.RESULTSThe fagellin enhanced the immunogenicity of Bap causingan increase in specific IgG titers in serum （P 〈 0.001）.Internal organs, i.e. , liver, lung and spleen of the Bap-Flagellin immunized group challenged with A. baumanniishowed significantly lower bacterial load compared tothe control group. The bacterial loads were studied ininternal organs. A. baumannii infected immunized animalswith Bap-Flagellin exhibited internal organs with minorbacterial load while P. aeruginosa PAO1 infected groupshowed heavy bacterial load of （4.3 ± 0.12） × 106,（1.1 ± 0.01） × 106 and （2.2 ± 0.22） × 106 per gram oflungs, liver and spleen respectively. Bacterial loads weredetected per gram of lungs, liver and spleen of the micegroup immunized with Bap were （1.2 ± 0.06） × 107, （11.1± 0.041） × 105 and （3.6 ± 0.42） × 106 respectively. In vivo neutralization assay indicated that all experimental mice groups, except for Flagellin administered group was signifcantly （ P 〈 0.05） protected against A. baumannii .CONCLUSIONThese results demonstrate that P. aeruginosa Flagellin as an adjuvant for BapA. baumannii could be a useful model to evaluate new vaccine against A. baumannii .
基金
Center for Molecular Microbiology, Shahed University for their financial support toward his work
