Unknown,Transcriptomics,Genomics,Proteomics

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Alanine scan of 11 amino acid long novel linear epitope from C.jejuni cj0669 protein [2]

ABSTRACT: A short sequence of 11 amino acids belonging to the cj0669 protein from Campylobacter jejuni NCTC 11168 which was previously identified as potentially immunogenig 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 seperated into three incubation chambers by the ProPlate 3-well module (Grace Biolabs) to allow for incubation with different antibodies in parallel. For specific interaction rabbit polyclonal IgG to C.jejuni was used, while non-specific binding to the epitope sequence was checked using rabbit polyclonal IgG to H. pylori (Abcam ab20459).

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: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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Alanine scan of 11 amino acid long novel linear epitope from C.jejuni cj0669 protein [2]

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