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Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1

View Article: PubMed Central - PubMed

ABSTRACT

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

No MeSH data available.


A Venn diagram to show the number of the total and overlapping differential protein profiles in different treatments. The number of total differential expression proteins and shared differential expression proteins from current work was displayed in each group. The overlapping differential proteins were obtained by comparing data from the three different groups.
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ijms-17-01419-f002: A Venn diagram to show the number of the total and overlapping differential protein profiles in different treatments. The number of total differential expression proteins and shared differential expression proteins from current work was displayed in each group. The overlapping differential proteins were obtained by comparing data from the three different groups.

Mentions: A total of 4748 unique proteins were identified with 95% confidence by the ProteinPilot search algorithm against the IPI (international protein index) mouse protein database v3.49. In order to evaluate as many differential expression proteins as possible, a strict cutoff value of a ≥1.50-fold or ≤0.5-fold change resulted in a final set of 3666 differential proteins in three groups. Among of them, 1288 differential proteins were identified in group LTB (p ≤ 0.05); 1640 differential proteins in group EVP1 (p ≤ 0.05); and 738 differential proteins in group LTB plus EVP1 treatment (p ≤ 0.05), respectively (Figure 2). The group LTB and group EVP1 had 1040 overlapping differential proteins, the group LTB and group LTB plus EVP1 had 316 overlapping differential proteins, and the group EVP1 and group LTB plus EVP1 had 469 overlapping differential proteins. There were 320 differential proteins overlapping among the three groups (Figure 2). The results indicated that the LTB (mucosal adjuvant) and EVP1 (antigen) coaction resulted in the reduction of the total differential protein expression in mø264.7.


Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1
A Venn diagram to show the number of the total and overlapping differential protein profiles in different treatments. The number of total differential expression proteins and shared differential expression proteins from current work was displayed in each group. The overlapping differential proteins were obtained by comparing data from the three different groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037698&req=5

ijms-17-01419-f002: A Venn diagram to show the number of the total and overlapping differential protein profiles in different treatments. The number of total differential expression proteins and shared differential expression proteins from current work was displayed in each group. The overlapping differential proteins were obtained by comparing data from the three different groups.
Mentions: A total of 4748 unique proteins were identified with 95% confidence by the ProteinPilot search algorithm against the IPI (international protein index) mouse protein database v3.49. In order to evaluate as many differential expression proteins as possible, a strict cutoff value of a ≥1.50-fold or ≤0.5-fold change resulted in a final set of 3666 differential proteins in three groups. Among of them, 1288 differential proteins were identified in group LTB (p ≤ 0.05); 1640 differential proteins in group EVP1 (p ≤ 0.05); and 738 differential proteins in group LTB plus EVP1 treatment (p ≤ 0.05), respectively (Figure 2). The group LTB and group EVP1 had 1040 overlapping differential proteins, the group LTB and group LTB plus EVP1 had 316 overlapping differential proteins, and the group EVP1 and group LTB plus EVP1 had 469 overlapping differential proteins. There were 320 differential proteins overlapping among the three groups (Figure 2). The results indicated that the LTB (mucosal adjuvant) and EVP1 (antigen) coaction resulted in the reduction of the total differential protein expression in mø264.7.

View Article: PubMed Central - PubMed

ABSTRACT

The nontoxic heat-labile toxin (LT) B subunit (LTB) was used as mucosal adjuvant experimentally. However, the mechanism of LTB adjuvant was still unclear. The LTB and enterovirus 71 (EV71) VP1 subunit (EVP1) were constructed in pET32 and expressed in E. coli BL21, respectively. The immunogenicity of purified EVP1 and the adjuvanticity of LTB were evaluated via intranasal immunization EVP1 plus LTB in Balb/c mice. In order to elucidate the proteome change triggered by the adjuvant of LTB, the proteomic profiles of LTB, EVP1, and LTB plus EVP1 were quantitatively analyzed by iTRAQ-LC-MS/MS (isobaric tags for relative and absolute quantitation; liquid chromatography-tandem mass spectrometry) in murine macrophage RAW264.7. The proteomic data were analyzed by bioinformatics and validated by western blot analysis. The predicted protein interactions were confirmed using LTB pull-down and the LTB processing pathway was validated by confocal microscopy. The results showed that LTB significantly boosted EVP1 specific systematic and mucosal antibodies. A total of 3666 differential proteins were identified in the three groups. Pathway enrichment of proteomic data predicted that LTB upregulated the specific and dominant MAPK (mitogen-activated protein kinase) signaling pathway and the protein processing in endoplasmic reticulum (PPER) pathway, whereas LTB or EVP1 did not significantly upregulate these two signaling pathways. Confocal microscopy and LTB pull-down assays confirmed that the LTB adjuvant was endocytosed and processed through endocytosis (ENS)-lysosomal-endoplasmic reticulum (ER) system.

No MeSH data available.