Immunogenic Compositions of Staphylococcus Epidermidis Polypeptide Antigens
a technology of staphylococcus epidermidis and compositions, applied in the field of immunogenic compositions, can solve the problems of difficulty in treating infections and necessitating difficulty in treating infections such as infections
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example 1
Bacterial Growth in 70% Serum
[0161]The following examples were performed using the clinical isolate Staphylcoccus epidermidis 0-47. The unannotated genomic sequence was available for this isolate from Incyte Corporation of Palo Alto, Calif. See Heilmann, C., et al., Infect Immun, 64(1): p. 277-82 (1996). To stimulate the expression of proteins, which may be clinically relevant to pathogenicity, cultures of bacteria were grown overnight in either 100% tryptic soy broth (TSB) or 70: 30 rabbit serum:TSB with shaking (200 rpm) at 37° C. The rabbit serum was obtained from Life Technologies, Rockville, Md. Bacteria were diluted from an overnight culture to an OD600˜0.1 and grown for 4 h until mid log phase. At mid log phase, the cells were harvested by centrifugation and further processed as described in the following examples.
example 2
Preparation Of Cell Wall Fractions for 2-D Gel Electrophoresis
[0162]The cell walls of Staphylcoccus epidermidis 0-47 grown as described in Example 1 were isolated and then prepared for two-dimensional gel electrophoresis. Bacterial pellets were resuspended to an OD600˜20 and washed twice with rocking for 15 minutes at 4° C. using Tris buffered saline (TBS, 20 mM Tris, pH 8.0, 150 mM NaCl). Serum proteins bound to the surface of the bacteria were removed by washing for 15 minutes at 4° C. with 20 mM Tris, pH 8.0 containing 1M NaCl. Bacteria grown in TSB were treated in the same manner as the bacteria grown in serum. The bacteria were again pelleted by centrifugation. To create protoplasts, the bacteria were then resuspended to OD600˜40 in TBS containing 30% sucrose, 100 μg / ml lysostaphin, 10 μg / ml DNase, 1 μg / ml Pefablock (Boehringer Mannheim, Indianapolis, Ind.), 10 μg / ml lysozyme and 100 units / ml mutanolysin and incubated at 37° C. for 1 hour. The resulting protoplasts were pellete...
example 3
Binding of Immune Serum and Biotinylated Serum Proteins to Cell Wall Proteins
[0167]After completion of the first and second dimensions of electrophoresis, the protein content of the gels was transferred onto nitrocellulose for binding assays. Specifically, the protein content of the gels was electro-blotted to nitrocellulose membranes (BioRad) using a semi-dry blotting apparatus (Owl Separations Systems, Portsmouth, N.H.) at 12V for 1 hour. The protein containing nitrocellulose membranes (blots) were then stained with Sypro Ruby protein blot stain (BioRad) following the manufacturer's instructions and visualized in a Fluor S Imager (BioRad). Each blot was incubated in blocking buffer (PBS with 0.05% Tween 20 and 5% dry milk) for 10 minutes at room temperature then incubated overnight with either a 1:2000 dilution of immune sera (Western blot) or 40 μg / ml biotinylated serum proteins (see below). Following overnight incubation, blots were washed 3× with wash buffer (PBS with 0.5% Twee...
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