Date

2014

Author

Çiçek, Mustafa

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Bordetella pertussis is a gram negative coccobacillus that causes pertussis known as whooping cough. After mass-vaccination started in 1940s, incidence of the disease has decreased. However, B. pertussis circulation in population has not been prevented completely. Starting from the first vaccination, development of several vaccines have been performed. These whole cell (Pw) and acellular pertussis (Pa) vaccines are not completely effective in terms of sustained, lifelong immunity and thus failure in eliminating subclinical infections poses a threat for both unimmunized infants and adults. The requirements for more effective acellular pertussis vaccines with protective proteins have raised the number of the studies to develop vaccines with high immune protective capacities. Recently, a surface antigen, namely glutamine-binding periplasmic protein GnlH was found to be among novel immunogenic proteins as shown by our immunoproteome group. Besides; another surface antigen putative peptidoglycan binding protein BP0020 was shown to include Lysm domain which is found in some of protective immunogens. These proteins were chosen as novel recombinant vaccine candidates. In the present study these proteins were tested for their immunoprotective capacity in mouse model. In order to stimulate humoral and cellular responses against B. pertussiss infection, the genes coding for gnlH and BP0020 proteins were amplified from the genomic DNA of B. pertussis strains and cloned into pGEM®-T Easy vector and sequenced. For their expression in Escherichia coli BL21(DE3) cells, expression vector pET-28a (+) was used together with IPTG induction system. After the expression of the desired proteins His-tag affinity chromatography together with dialysis was used for the purification of the proteins. Following Western blot analysis, 10 µg of both GlnlH and BP0020 proteins were used to immunize BALB/c mice (16-18 g) at days 0 and 21. B. pertussis Saadet live cells were then administered intranasally to challenge mice. Bacterial colonization in mice was determined after the removal of the lungs at days 5 and 8. When compared to the control groups, bacterial colonization were found to be decreased in the lungs of the mice immunized with 10 µg recombinant GlnH and BP0020 proteins. ELISA for serum-specific IgG levels was performed after the collection of sera. The results showed that IgG levels were significantly higher in immunized mice. In addition; serum IFN-γ levels were found to be higher in vaccinated mice in comparision to control groups.

Subject Keywords

Whooping cough., Bordetella pertussis., Vaccines., Glutamine., Peptidoglycans., Recombinant proteins., Mice as laboratory animals.

URI

http://etd.lib.metu.edu.tr/upload/12617009/index.pdf
https://hdl.handle.net/11511/23451

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Graduate School of Natural and Applied Sciences, Thesis