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Screening for novel immungenic proteins of Klebsiella pneumoniae MGH 78578

ABSTRACT: The screening of a cDNA derived expression library of Klebsiella pneumoniae MGH 78578 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 (ompA, mdh) and negative (pyrC, gapA) reference proteins. The goal of the screening was to identify potential novel immunogenic proteins from K. pneumoniae 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, 14 novel immunogenic proteins could be identified. In total 1536 (4 x 384) 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: ompA1, ompA2 and mdh (3 x 40 replicates) as positive reference proteins, as they have been described as immunogenic before. gapA and pyrC (2 x 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 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). For identification rabbit polyclonal antibody to K. pneumoniae (Acris AP00792PU-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.

ORGANISM(S): Klebsiella pneumoniae

SUBMITTER: Sebastian Hoppe

PROVIDER: E-GEOD-52536 | 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.

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.

Project description:Detection of immunogenic proteins remains an important task for life sciences as it nourishes the understanding of pathogenicity, illuminates new potential vaccine candidates and broadens the spectrum of biomarkers applicable in diagnostic tools. Traditionally, immunoscreenings of expression libraries via polyclonal sera on nitrocellulose membranes or screenings of whole proteome lysates in 2-D gel electrophoresis are performed. However, these methods feature some rather inconvenient disadvantages. Screening of expression libraries to expose novel antigens from bacteria often lead to an abundance of false positive signals owing to the high cross reactivity of polyclonal antibodies towards the proteins of the expression host. A method is presented that overcomes many disadvantages of the old procedures. We incorporated a fusion tag prior to our genes of interest and attached the expressed fusion proteins covalently on microarrays. This enhances the specific binding of the proteins compared to nitrocellulose. Thus, it helps to reduce the number of false positives significantly. It enables us to screen for immunogenic proteins in a shorter time, with more samples and statistical reliability. We validated our method by employing several known genes from Campylobacter jejuni NCTC 11168. Four of proteins that have previously been described as immunogenic have successfully been assessed immunogenic abilities with our method. One protein with no known immunogenic behaviour before suggested potential immunogenicity. The method presented offers a new approach for screening of bacterial expression libraries to illuminate novel proteins with immunogenic features. It could provide a powerful and attractive alternative to existing methods and help to detect and identify bacterial virulence factors, vaccine candidates and potential biomarkers. The overall study was done with five technical replicates. Ten proteins derived from Campylobacter jejuni were tested regarding their immunogenic potential. The ten proteins derived from Campylobacter jejuni were fusion proteins, i.e., N-terminal HaloTag fused to: Gene <--> Protein cjaA <--> putative amino-acid transporter periplasmic solute-binding protein hisJ <--> histidine-binding protein precursor pal <--> peptidoglycan associated lipoprotein flaC <--> flagellin flaA <--> flagellin peb1a <--> bifunctional adhesin/ABC transporter aspartate/glutamate-binding protein pyrC <--> dihydroorotase (EC:3.5.2.3) pseB <--> UDP-GlcNAc-specific C4,6 dehydratase/C5 epimerase gapA <--> glyceraldehyde 3-phosphate dehydrogenase (EC:1.2.1.12) argC <--> N-acetyl-gamma-glutamyl-phosphate reductase (EC:1.2.1.38)

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Screening for novel immungenic proteins of Klebsiella pneumoniae MGH 78578

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