Clostridium difficile polypeptides as vaccine
A technology of Clostridium difficile and polypeptide fragments, applied in the field of polypeptides of Clostridium difficile, can solve problems such as the inability to completely prevent colonization pathogens
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Embodiment 1
[0240] Example 1: Identification of Clostridium difficile polypeptides
[0241] To identify polypeptides for use in vaccines against C. difficile, the inventors analyzed the repertoire of approximately 3780 predicted polypeptides encoded by C. difficile 630 using the PSORT program (220). The identified protein has a predicted peripheral subcellular localization. Specifically, the following groups were identified:
[0242] • Cell wall-associated proteins, identified by the presence or absence of a typical LPXTG-like motif representing the site that attaches the protein to the outside of the bacterial cell wall.
[0243] • Extracellular proteins, identified by the presence of an N-terminal leader peptide (which normally directs the protein product to the extracellular matrix) and / or by sequence similarity to other bacterial proteins known to be exported.
[0244] • Proteins with bacterial surface localization.
[0245] Other polypeptides are selected based on their sequence...
Embodiment 2
[0246] Example 2: Identification of secreted C. difficile polypeptides
[0247]C. difficile secretome preparations were performed as follows: C. difficile (strains 630 and Stoke Mandeville) were grown in brain heart infusion (BHI) or chemically defined medium (CDM, M.N. Mickelson, J. of Bact., 1964, 88:158-164). Bacteria were plated overnight on agar plates and subsequently grown anaerobically in 100 mL of the appropriate medium at 37 °C until mid-log phase (OD 600 0.5) or early stable period (OD 600 is 0.9). Bacteria were removed by centrifugation at 3,500 xg for 10 minutes at 4°C and the supernatant was filtered through a 0.22 μm pore size filter (Millipore). Proteins in the supernatant were precipitated overnight using 10% w / v trichloroacetic acid, 0.04% w / v sodium deoxycholate. Resuspend the protein in a solution containing 5mM dithiothreitol, 0.1% (Waters) in 50 mM ammonium bicarbonate, heated at 90° C. for 10 minutes and digested overnight with 2 ug of trypsin (P...
Embodiment 3
[0252] Example 3: Analysis of cell lysates by NMR
[0253] Bacterial cells carrying vectors for recombinant protein expression were initially washed by resuspending in 800 uL M9 1X buffer (pH 7.4), centrifuging (5 min at 3,000 rpm) and discarding the supernatant. The remaining bacterial pellet was resuspended in 600 uL of M9 buffer, and cells were lysed by sonication followed by centrifugation at 4°C (14,000 rpm, 20 minutes). The supernatant was then isolated for NMR analysis. 10% D2O (by volume) was added to the samples for NMR analysis. Use 900 or 800MHz NMR to acquire fast HSQC or TROSY experiments and process through topspin software ( image 3 ).
[0254] In NMR analysis, the HSQC spectrum of Dif44 shows a good dispersion of pictures (pics), where Figure 4 Roughly equal intensities in show that Dif44 is well folded. The results of NMR analysis of cell lysates are summarized in figure 2 . These results identify polypeptides with a propensity to stably maintain t...
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