Expression, Purification and Characterization of Three Overlapping Immunodominant Recombinant Fragments from Bordetella pertussis Filamentous Hemagglutinin

Abstract

Background: Filamentous hemagglutinin (FHA) is one of the most important immunoprotective antigens of Bordetella pertussis (B. pertussis) and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated.

Methods: Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli (E. coli) BL21(DE3) strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells (PBMC) proliferation and IFN-γ production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively.

Results: Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21(DE3). SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN-γ production.

Conclusion: Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immuno-protective.

Keywords: Bordetella pertussis; Filamentous hemagglutinin; Immunodominant; Prokaryotic expression; Recombinant antigen.

Figure 1

Schematic representation of precursor FHA, mature native FHA and the location of the three overlapping recombinant FHA fragments (rFHA1-3) employed in this study. The amino acid lengths and positions of all proteins are shown in parentheses

Figure 2

Representative results of PCR amplification; A) and restriction enzyme digestion; B) of the three FHA coding sequences. Agarose gel electrophoresis of amplified PCR products of rFHA1-3 fragments confirms their 1152, 1119 and 1119 bp sizes, respectively. Double digestion of pET2 2b(+)-rFHA1-3 constructs with EcoRI and HindIII endonucleases indicates the proper insertion of rFHA1-3 DNA segments into the expression vector. SM: DNA size marker

Figure 3

Induction of rFHA1; A), rFHA2; B) and rFHA3; C) proteins expression in E. coli BL21 (DE3). IPTG was added to a logarithmic liquid culture of transformed bacteria at 1 mM concentration when OD_600nm was 0.6. Pre-induction and post-induction samples were collected at different time points and run on 12% SDS-PAGE followed by Coomassie blue staining. The arrow in the middle of the gel shows the expected target protein molecular weight (∼ 40 kDa); SM: protein size marker

Figure 4

Analysis of the purified recombinant FHA fragments by SDS-PAGE; A) and Western blotting; C). All proteins were purified through His-tag by Ni-NTA column. SDS-PAGE analysis was performed using a 12% polyacrylamide gel followed by Coomassie blue staining. Western blot analyses of rFHA1-3 fragments were performed using two different preparations of detecting antibodies, including rabbit anti-FHA produced in our lab and a commercial rabbit anti-His tag. SM: protein size marker

Figure 5

In vitro cell proliferation; A) and IFN-γ production; B) induced by native and recombinant FHA proteins. Peripheral blood mononuclear cells (PBMC) were collected from four healthy adults before and at 1 and 4 weeks after vaccination with a commercial acellular pertussis vaccine. 2*10⁵ cells were stimulated with appropriate concentrations of native commercial FHA (2 µg/ml), three recombinant FHA fragments (5 µg/ml) and PHA (5 µg/ml). After 2 days stimulation for PHA and 6 days stimulation for FHA antigens, H3-Thymidine was added and the cells were harvested after 18 hours stimulation. Stimulation indices (SI) were calculated by dividing the mean counts per minute (CPM) of cells exposed to the FHA or PHA to the mean CPM of unstimulated cells incubated with culture medium alone. For IFN-γ assay, culture supernatants were harvested 48 hours after stimulation. For both proliferation and IFN-γ production, mean values ± SEM obtained from four subjects are indicated