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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.


Western blot analysis of some differential proteins. The target proteins of LTB treated mouse mø264.7 was expressed in line with the trend of iTRAQ-MS analysis results. The area value of protein bands was measured by Image-pro plus tools and the proteins change significantly (* p < 0.05). More than three biological replicates were performed.
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ijms-17-01419-f006: Western blot analysis of some differential proteins. The target proteins of LTB treated mouse mø264.7 was expressed in line with the trend of iTRAQ-MS analysis results. The area value of protein bands was measured by Image-pro plus tools and the proteins change significantly (* p < 0.05). More than three biological replicates were performed.

Mentions: To test the validity of iTRAQ-LC-MS/MS data, the western blot was performed and the results proved that the expression of the differential proteins (EDIL3, STAT1, HSPA4, GSTO1, PRDX1) was consistent with the data obtained from iTRAQ (repeats > 3, Figure 6). These proteins were classified as immune system process proteins by PANTHER. The results validated the compounds identified in Table 1.


Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1
Western blot analysis of some differential proteins. The target proteins of LTB treated mouse mø264.7 was expressed in line with the trend of iTRAQ-MS analysis results. The area value of protein bands was measured by Image-pro plus tools and the proteins change significantly (* p < 0.05). More than three biological replicates were performed.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01419-f006: Western blot analysis of some differential proteins. The target proteins of LTB treated mouse mø264.7 was expressed in line with the trend of iTRAQ-MS analysis results. The area value of protein bands was measured by Image-pro plus tools and the proteins change significantly (* p < 0.05). More than three biological replicates were performed.
Mentions: To test the validity of iTRAQ-LC-MS/MS data, the western blot was performed and the results proved that the expression of the differential proteins (EDIL3, STAT1, HSPA4, GSTO1, PRDX1) was consistent with the data obtained from iTRAQ (repeats > 3, Figure 6). These proteins were classified as immune system process proteins by PANTHER. The results validated the compounds identified in Table 1.

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.