Date

2011

Author

Yılmaz, Çiğdem

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Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. It becomes widespread among adolescent and adults as well as infants. Although availability of effective pertussis vaccines seems to decrease the incidence of the disease, B. pertussis circulation in population has not been eliminated. It is thought that the antigenic drifts in major protective antigens and continued circulation of B. pertussis strains will result in gradual loss of the efficacy of the current pertussis vaccines. Therefore, development of more effective acellular pertussis vaccines with conserved protective proteins is a convenient strategy to provide a better protection against whooping cough. In this study, immune responses against putative peptidyl-prolyl cis-trans isomerase (PPIase) which was shown to be immunogenic in B. pertussis for the first time by our immunoproteome group and FimX whose expression was found higher in our local Saadet strain were determined in mice. The genes encoding FimX and putative PPIase were amplified by PCR, cloned into pGEM®-T Easy vector and sequenced. The genes were then introduced into pET-28a (+) vector and they were expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins were purified by His-tag affinity chromatography and dialyzed. After Western blot analyses, 20 µg and 80 µg recombinant FimX and 80 µg recombinant putative PPIase were used to immunize BALB/c mice (16-18 g) at day 0 and 21. The mice were challenged intranasally with 2.5 x 109 live B. pertussis Saadet cells. Before second immunization and challenge, the sera were collected to carry out ELISA for measurement of serum-specific IgG levels. According to ELISA results, IgG levels in the mice immunized with 20 µg and 80 µg recombinant FimX were found significantly higher than in control groups at both first and second vaccinations (p<0.01). On the other hand, immunization with 160 µg recombinant putative PPIase provided a significant increase in IgG level (p<0.05) only at second vaccination. The lungs of the mice were removed at day 2, 5, 8 after challenge and bacterial colonization was determined. No significant decrease in bacterial colonization was observed in the lungs of the mice immunized with 20 µg and 80 µg recombinant FimX and 80 µg recombinant putative PPIase with respect to control groups. After respiratory challenge and second immunization (at day 30) with 20 µg and 80 µg recombinant FimX, the spleens of the mice were removed and a spleen cell culture was obtained. Supernatants were collected after induction of the cells with the recombinant protein and cytokine ELISA was carried out to measure IFN-γ level. No significant difference was observed between control and vaccinated mice in terms of IFN-γ production.

Subject Keywords

Bordetella pertussis., Pertussis vaccines., Whooping cough

URI

http://etd.lib.metu.edu.tr/upload/12613684/index.pdf
https://hdl.handle.net/11511/21258

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Graduate School of Natural and Applied Sciences, Thesis