Alanine scan of 11 amino acid long novel linear epitope from C.jejuni cj0669 protein [2]
Ontology highlight
ABSTRACT:
ORGANISM(S): Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819
SUBMITTER: Sebastian Hoppe
PROVIDER: E-GEOD-46651 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168, which was previously identified as potentially immunogenic, was analyzed via alanine scanning to narrow down the significant amino acid residues within the sequence. Twelve peptides, one representing the original sequence and eleven peptides with each residue replaced by alanine in turn, were synthesized on microarrays by JPTs Pepstar Technology. For each microarray, nine replicates for each peptide were spotted. The microarray was separated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interactions, rabbit polyclonal IgG to C. jejuni was used, while unspecific binding to the epitope sequence was checked using rabbit polyclonal IgG to Salmonella enterica.
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Project description:A number of seven proteins were selected during immunoscreening and further analyses. The proteins were in silico divided into overlapping 15-mer oligopeptides with an overlap of 11 residues. The microarrays were incubated with different antibodies to C. jejuni, Escherichia coli and Salmonella enterica. Each microarray was separated into three individual incubation chambers using ProPlate 3-Well modules. Within each incubation chamber, each peptide was spotted in triplicate with the controls spotted nine times each. The controls included human-IgG, rabbit-IgG, mouse-IgG and myelin basal protein (MBP). Each chamber was incubated independently using different polyclonal antibodies to C. jejuni, and for specificity testing, with an antibody to E. coli or S. enterica. Thus, samples 4_1, 4_2, 5_1 and 5_2 represent epitope mapping of three proteins with C. jejuni antibodies, while 6_1, 6_2, 7_1 and 7_2 represent the data after incubation with an E. coli antibody investigating unspecific interactions of the antibody to the potential linear epitopes from C. jejuni. Finally, for four different proteins from C. jejuni, the set two indicated by S2 was performed. Here, S2_6_1, S2_7_1, S2_7_2, S2_8_1 and S2_8_2 indicate epitope mapping after incubation with antibodies to C. jejuni, while the remaining samples were performed to test these latter 4 proteins for specificity by incubation with antibody to S. enterica.
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Project description:A number of six proteins were selected during immunoscreening and further analyses. The proteins were divided in silico into overlapping 15-mer oligopeptides with an overlap of 11 residues. The microarrays were incubated with different antibodies to K. pneumoniae, C. jejuni and S. aureus. Each microarray was seperated into three individual incubation chambers using ProPlate 3-Well modules. Within each incubation chamber each peptide was spotted in triplicate with the controls spotted nine times each. The controls included Human-IgG, Rabbit-IgG, Mouse-IgG and Myelin Basal Protein. Each chamber was incubated independently using different polyclonal antibodies to K. pneumoniae and for specificity testing with antibodies to C. jejuni or S. aureus. Thus, samples 2_1, 2_2, 3_1 and 3_2 represent epitope mapping of three proteins and S2_5_1, S2_5_2, S2_6_1 and S2_6_2 epitope mapping of three different proteins with K. pneumoniae antibodies, while 4_1, 4_2, 5_1 and 5_2 represent specificity assays for the first set of proteins (using C. jejuni and S. aureus antibodies) and S2_1_1, S2_1_2, S2_2_1, S2_2_2 (C. jejuni AB) as well as S2_7_1, S2_7_2, S2_8_1, S2_8_2 (S. aureus AB) for the second set of proteins.
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Project description:Screening of 22 novel proteins derived from Campylobacter jejuni NCTC 11168 identified prior via screening of cDNA libraries. The full-length proteins were attached using a specific HaloTag to their corresponding ligand surface, HaloLink. Screening was performed using three different polyclonal antibodies to Campylobacter jejuni and detection was achieved by goat polyclonal antibody to rabbit IgG conjugated with Chromeo-546. In order to assess their potential immungenic nature and rank the proteins investigated, comparative analysis using already described antigens from C. jejuni were used in the assay. Each microarray was separated into different incubation chambers using the ProPlate (Grace Biolabs) multi-well gaskets. While for two slides (2009 and 2447), three chambers were used, the remaining slides were designed to use 16 different compartments. Each compartment could be incubated with different antibodies and represent individual replicates of the slides. As positive references, hisJ and cjaA were used. For negative controls, argC and gapA were used, and the crude lysates of the expression host (Acella E. coli) and buffer were spotted as well. For slides 2009 and 2447, three-well gaskets were used allowing for seven replicates per sample, while only incubation with one antibody. Slides 416033 and 416826 used 16-well gaskets and only hisJ and argC as protein references. Samples and controls were spotted in quadruplicate. Finally, for 1000 and 1001, samples were spotted in triplicate, whereas controls were spotted in quadruplicate using hisJ, cjaA, argC and gapA as protein references.
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Project description: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. 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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Project description: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. 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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