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| Status |
Public on Apr 06, 2013 |
| Title |
Screening for novel immunogenic proteins of Campylobacter jejuni NCTC 11168 |
| Organism |
Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819 |
| Experiment type |
Protein profiling by protein array
|
| Summary |
The screening of a cDNA derived expression library of Campylobacter jejuni NCTC 11168 expressed in E. coli using a fusion construct and specific HaloTag interaction to a modified surface is shown. 1536 different clones were screened including positive (hisJ, cjaA, peb1a) and negative (argC, pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from C. jejuni by selecting clones showing a high signal intensity in comparison to the known antigens used as positive markers. Afterwards, the most promising clones were sequenced to identify the gene and corresponding protein, and these proteins were then investigated further. Consequently, 22 novel immunogenic proteins could be identified.
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| Overall design |
In total, 1536 (4 x 384) different lysates were spotted on different microarray slides. Each slide contained 3600 distinct spots, separated 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: hisJ, cjaA and peb1 (3 x 40 replicates) as positive reference proteins, as they have been described as immunogenic before; argC and pyrC (2 x 40 replicates) as negative reference proteins; two sets of E. coli cell lysates without fusion proteins expressed, namely Acella electrocompetent cells (40 replicates) and KRX (32 replicates); and a buffer control (24 replicates). Therefore, each set of replicate slides contained 376 different samples and 8 controls. 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 C. jejuni (Acris AP24002PU-N) 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(s) |
23734261 |
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| Submission date |
Mar 18, 2013 |
| Last update date |
Jul 09, 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 (4)
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| GPL16810 |
IBMT Campylobacter jejuni NCTC 11168 1.5k protein screening array (1 of 4) |
| GPL16811 |
IBMT Campylobacter jejuni NCTC 11168 1.5k protein screening array (2 of 4) |
| GPL16812 |
IBMT Campylobacter jejuni NCTC 11168 1.5k protein screening array (3 of 4) |
| GPL16813 |
IBMT Campylobacter jejuni NCTC 11168 1.5k protein screening array (4 of 4) |
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| Samples (12)
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| GSM1100457 |
Immunoscreening C. jejuni 1, replicate 1 |
| GSM1100458 |
Immunoscreening C. jejuni 1, replicate 2 |
| GSM1100459 |
Immunoscreening C. jejuni 1, replicate 3 |
| GSM1100460 |
Immunoscreening C. jejuni 2, replicate 1 |
| GSM1100461 |
Immunoscreening C. jejuni 2, replicate 2 |
| GSM1100462 |
Immunoscreening C. jejuni 2, replicate 3 |
| GSM1100463 |
Immunoscreening C. jejuni 3, replicate 1 |
| GSM1100464 |
Immunoscreening C. jejuni 3, replicate 2 |
| GSM1100465 |
Immunoscreening C. jejuni 3, replicate 3 |
| GSM1100466 |
Immunoscreening C. jejuni 4, replicate 1 |
| GSM1100467 |
Immunoscreening C. jejuni 4, replicate 2 |
| GSM1100468 |
Immunoscreening C. jejuni 4, replicate 3 |
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| This SubSeries is part of SuperSeries: |
| GSE44717 |
Identification of novel immunodominant proteins from Campylobacter jejuni and the characterization of linear epitopes |
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| Relations |
| BioProject |
PRJNA193420 |
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