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Microarray-based method for screening of immunogenic proteins from bacteria.

Hoppe S, Bier FF, von Nickisch-Rosenegk M - J Nanobiotechnology (2012)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam, Am M├╝hlenberg 13, 14476 Potsdam, Germany. sebastian.hoppe@ibmt.fraunhofer.de

ABSTRACT

Background: 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.

Results: Four 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.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.

Conclusions: 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 vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

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Agarose gel of PCR products after amplification of Campylobacter jejuni genes from genomic DNA. The band sizes match the respective length of each gene. Refer to Table 2 for expected gene lengths. As markers Hyper Ladder I (M) and II (M2) were used both supplied by Bioline.
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Figure 3: Agarose gel of PCR products after amplification of Campylobacter jejuni genes from genomic DNA. The band sizes match the respective length of each gene. Refer to Table 2 for expected gene lengths. As markers Hyper Ladder I (M) and II (M2) were used both supplied by Bioline.

Mentions: We successfully amplified all but one gene (cfrA) from the genomic DNA of Camyplobacter jejuni. All eleven genes that were successfully amplified showed the correct length (see Figure 3) and were subsequently cloned to KRX single-step competent cells. The correct insert size was determined by Colony PCR. Plasmids from clones containing the correct-sized inserts were isolated and the MCS was sequenced using both a forward and a reverse primer (HT7 For and Flexi R). During cloning an extra GTT, which encodes for a valine residue in the proteins' primary structures, was inserted immediately prior to each stop codon. As a universal stop codon TAA was used, replacing other stop codons if present. This was mainly done to gain maximum flexibility during cloning as the Flexi vector system enables the direct transfer to other vectors with different tags. A GTT is mandatory to later transfer the gene of interest to a vector encoding a C-terminal tag.


Microarray-based method for screening of immunogenic proteins from bacteria.

Hoppe S, Bier FF, von Nickisch-Rosenegk M - J Nanobiotechnology (2012)

Agarose gel of PCR products after amplification of Campylobacter jejuni genes from genomic DNA. The band sizes match the respective length of each gene. Refer to Table 2 for expected gene lengths. As markers Hyper Ladder I (M) and II (M2) were used both supplied by Bioline.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368735&req=5

Figure 3: Agarose gel of PCR products after amplification of Campylobacter jejuni genes from genomic DNA. The band sizes match the respective length of each gene. Refer to Table 2 for expected gene lengths. As markers Hyper Ladder I (M) and II (M2) were used both supplied by Bioline.
Mentions: We successfully amplified all but one gene (cfrA) from the genomic DNA of Camyplobacter jejuni. All eleven genes that were successfully amplified showed the correct length (see Figure 3) and were subsequently cloned to KRX single-step competent cells. The correct insert size was determined by Colony PCR. Plasmids from clones containing the correct-sized inserts were isolated and the MCS was sequenced using both a forward and a reverse primer (HT7 For and Flexi R). During cloning an extra GTT, which encodes for a valine residue in the proteins' primary structures, was inserted immediately prior to each stop codon. As a universal stop codon TAA was used, replacing other stop codons if present. This was mainly done to gain maximum flexibility during cloning as the Flexi vector system enables the direct transfer to other vectors with different tags. A GTT is mandatory to later transfer the gene of interest to a vector encoding a C-terminal tag.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Fraunhofer Institute for Biomedical Engineering, Branch Potsdam, Am M├╝hlenberg 13, 14476 Potsdam, Germany. sebastian.hoppe@ibmt.fraunhofer.de

ABSTRACT

Background: 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.

Results: Four 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.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.

Conclusions: 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 vaccine candidates, biomarkers and potential virulence-associated factors with immunogenic behaviour furthering the knowledge of virulence and pathogenicity of studied bacteria.

Show MeSH
Related in: MedlinePlus