pertussis DNA using primers with the restriction sites BamHI (PrnProF-BamHI) and NdeI (PRNProR-NdeI). The plasmid pSKPD25FpPRN3 was cut with BamHI and NdeI to generate a fragment which had lost the FHA promoter. The PRN promoter Bcl-2 inhibitor was ligated in its place. After transformation into E. coli and verification by restriction analysis, the resulting plasmid was designated as pSKPD25PRN3 (Figure 5C). The plasmid was

cut with NotI and inserted into pSS4245 cut with the same enzyme. The resulting construct, pSSPD2prn was transferred into E. coli SM10 to conduct the allelic exchange. The resulting B. pertussis strain was designated as Bp-WWE. Integration of the prn gene at its designated position was confirmed by PCR with the primers that specifically bind only to the upstream 5′ (5′FPD2-int and PRNProR-NdeI primers), 3′ (PRNF-int and 3′RPD2-int primers) downstream flanking regions, and inside the prn gene. PT, FHA and PRN expression in shake flask culture The Bp-WWC, Bp-WWD and Bp-WWE strains were grown in shake flasks with 100 mL MSS medium supplemented with methylated β-cyclodextrin (1 g/l) at 35°C with shaking speed of 200 rpm. After 32-48 h of growth, the culture supernatants were collected and assayed by ELISA to quantify the PT and FHA expression level. As PRN releasing from its membrane-bound precursor is the result of an imprecise cleavage by unidentified proteases

[34], PRN expression was determined by Western blot with densitometric analysis to evaluate the integrity of the antigen. Selleckchem GW786034 Org 27569 This assay was conducted both on the clarified culture supernatant and the cell extract obtained by heating cell suspension in isotonic buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.002% NaN3, and 1 mM PMSF) at 60°C for 30 min and the supernatant was collected after centrifugation

at 10,000 × g, 4°C for 30 min. ELISA assay for PT and FHA Purified rabbit polyclonal antibodies against PT or FHA (NLAC, Thailand) with the dilution of 1:1000 in carbonate/bicarbonate buffer (pH 9.6) were coated in 96-well plates (NUNC Maxisorp, Denmark) for 100 μL per well and incubated overnight at 4°C. After 3 time-washing with phosphate-buffered selleckchem saline pH 7.4 containing 0.1% Tween 20 (PBST), blocking was performed using 100 μL per well of 3% bovine serum albumin (BSA)-PBST then incubated at 37°C for 1 h. After discarding the blocking buffer and washing, dilutions of the standard PT, FHA or samples were loaded and incubated at 37°C for 1 h. Then, anti-PT mouse monoclonal antibody (Abcam, USA) at 1:30,000 dilution or anti-FHA mouse monoclonal antibody (NIBSC, UK) at 1:10,000 dilution in blocking buffer was added and incubated under the same conditions. After washing the wells for three times with PBST, 100 μL of rabbit anti-mouse (H + L) IgG-HRP conjugate (Abcam, USA) in blocking buffer at 1:10,000 dilution was used as secondary antibody and incubated for 37°C for 1 h.