3 Material and Methods
urea for 2-D gel electrophoresis (2-DE). Protein concentration was determined using the
Bio-Rad DC assay in a 1:4 dilution according to the manufacturer’s instructions.
3.2.9 Subtractive proteome analysis (SUPRA)
2-DE was performed according to the method described by Bernardo et al. (2004) using
the Multiphor II system according to the manufacturer’s instructions. Proteins dissolved in
8 M urea were separated on 18-cm immobilised pH gradient (IPG) strips using non-linear
pH ranges of 3 to 10 and 4 to 7 (GE Healthcare). Isoelectric focusing was performed using
500 µg protein for Coomassie blue-stained gels and Western blots and 100 µg for silver-
stained gels and Western blots. S. aureus ACW-proteins were then separated on 12.5 %
Tris-glycine-SDS gels (25 cm by 20 cm by 1.0 mm) using the Ettan Dalt II system (GE
Healthcare).
For immunoblotting, proteins separated by 2-DE were transferred to nitrocellulose mem-
branes using a Trans-Blot cell (Bio-Rad) according to the manufacturer’s instructions. Mem-
branes were probed ON at 4 ° C either with IVIG at a dilution of 1:500 or with IVIG de-
pleted of S. aureus specific IgGs (dSaIVIG) using an equivalent antibody concentration.
Specific detection of immune complexes was performed using anti-human IgG-HRP con-
jugated secondary antibody at a 1:2,500 dilution. After treatment with dSaIVIG the mem-
brane was stripped at 50 ° C using stripping buffer containing 62.5 mM Tris-HCl (pH 6.8),
2 % SDS, and 100 mM β -mercaptoethanol and incubated with IVIG. To assess the repro-
ducibility of spot patterns, three separate experiments were performed. Signals of interest
were matched with corresponding spots on the Coomassie stained preparative gel, excised,
and digested with trypsin, followed by matrix-assisted laser desorption ionisation - time of
flight (MALDI-TOF) mass spectrometry performed as previously described (Bernardo et al.,
2004). Probability-based scoring (probability based on implementation of the Mowse algo-
rithm for assessing peptide and protein matches (Pappin et al., 1993)) was performed by
using the equation -10 x log(P), where P is the probability that the observed peptide match
is a random event. Scores greater than 56 were considered significant.
3.2.10 Cloning, expression and purification of vaccine candidates
The ORFs encoding BT1, BT2 and BT3, lacking potential secretion signalling sequences,
were amplified from S. aureus ATCC 29213 genomic DNA by PCR using Pfu-Polymerase.
The PCR products were purified using the QiaQuick PCR purification kit according to the
manufacturer’s instructions. PCR product and dephosphorylated pET29b vector were re-
stricted using NcoI and XhoI in 2x Tango buffer for 2 hours at 37 ° C, subsequently ligated
and transformed into E. coli DH5 α and selected on Kanamycin-LB plates. Vector pET29b
provides a N-terminal S-tag and a C-terminal His-tag in frame with the inserted gene. Cor-
rect constructs, identified by colony PCR and restriction analysis, were sequenced and
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