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| Status |
Public on Oct 01, 2013 |
| Title |
Screening for novel immungenic proteins of Salmonella Enteritidis 125109 |
| Platform organism |
Escherichia coli |
| Sample organism |
Salmonella enterica subsp. enterica serovar Enteritidis |
| Experiment type |
Protein profiling by protein array
|
| Summary |
The screening of a cDNA derived expression library of Salmonella Enteritidis 125109 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. Thus, 1536 different clones were screened including positive (fimA) and negative (argC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from S. Enteritidis by selecting clones showing a high signal intensity in comparison to the known antigens used as positve markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein and these proteins were then investigated further. Consequently, 9 novel immunogenic proteins could be identified.
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| Overall design |
In total 1536 different lysates were spotted on different microarray slides. Each slides contained 3600 distinct spots, seperated into two compartments of 1800 spots each. Each compartment comprised quadruplicate replicates of each sample lysate with controls being analysed with more replicates. As controls we used: fimA from 3 different samples (40 replicates) as positive reference proteins, as it has been described as immunogenic before. GapA from Klebsiella pneumoniae and Campylobacter jejuni (40 replicates) as negative reference proteins, additionally two sets of E.coli cell lysates without fusionprotein expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates). Last but not least a buffer control (24 replicates) was included. Therefore, each set of replicate slides contained 384 different samples. Each screening was performed with three replicate slides. Consequently, a total number of 12 slides were screened (4 sets of samples x 3 replicates each). For identification rabbit polyclonal antibody to S. enterica (Abcam ab35156) as primary and Goat polyclonal to Rabbit IgG conjugated with ChromeoTM-546 (Abcam ab60317) as secondary antibody were used. The top compartment was incubated first with primary antibody, while the bottom compartment was incubated with PBS at the same time. Afterwards both compartments were incubated with secondary antibody.
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| Citation missing |
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| Submission date |
Aug 06, 2013 |
| Last update date |
Oct 02, 2013 |
| Contact name |
Sebastian Hoppe |
| E-mail(s) |
sebastian.hoppe@izi-bb.fraunhofer.de
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| Organization name |
Fraunhofer Institute for Cell Therapy and Immunology
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| Department |
Bioanalytics and Biosensorics
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| Lab |
Molecular Bioengineering
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| Street address |
Am Mühlenberg 13
|
| City |
Potsdam |
| ZIP/Postal code |
14476 |
| Country |
Germany |
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| Platforms (1) |
| GPL17538 |
IBMT Salmonella Enteritidis library screening |
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| Samples (12)
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| GSM1202499 |
immunoscreening S. Enteritidis 1 (415842) |
| GSM1202500 |
immunoscreening S. Enteritidis 1 (415843) |
| GSM1202501 |
immunoscreening S. Enteritidis 1 (4169664) |
| GSM1202502 |
immunoscreening S. Enteritidis 2 (416927) |
| GSM1202503 |
immunoscreening S. Enteritidis 2 (416929) |
| GSM1202504 |
immunoscreening S. Enteritidis 2 (4169663) |
| GSM1202505 |
immunoscreening S. Enteritidis 3 (1075) |
| GSM1202506 |
immunoscreening S. Enteritidis 3 (1077) |
| GSM1202507 |
immunoscreening S. Enteritidis 3 (1970) |
| GSM1202508 |
immunoscreening S. Enteritidis 4 (1973) |
| GSM1202509 |
immunoscreening S. Enteritidis 4 (2500) |
| GSM1202510 |
immunoscreening S. Enteritidis 4 (2502) |
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| Relations |
| BioProject |
PRJNA214479 |
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