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Int. J. Mol. Sci. 2016, 17(9), 1419; doi:10.3390/ijms17091419

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

1
Key Laboratory of Biochemistry and Molecular Biology, The Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016, China
2
Institute of Life Science, Chongqing Medical University, Chongqing 400016, China
*
Author to whom correspondence should be addressed.
Academic Editor: Cheorl-Ho Kim
Received: 16 May 2016 / Revised: 17 August 2016 / Accepted: 23 August 2016 / Published: 27 August 2016
(This article belongs to the Collection Advances in Proteomic Research)
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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. View Full-Text
Keywords: proteome; iTRAQ; immune; mucosal adjuvant; enterovirus 71 VP1; ETEC (Enterotoxigenic Escherichia coli); LTB (labile toxin B subunit) proteome; iTRAQ; immune; mucosal adjuvant; enterovirus 71 VP1; ETEC (Enterotoxigenic Escherichia coli); LTB (labile toxin B subunit)
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Liu, L.; Ma, Y.; Zhou, H.; Wu, M. Quantitative Proteomic Analysis of Escherichia coli Heat-Labile Toxin B Subunit (LTB) with Enterovirus 71 (EV71) Subunit VP1. Int. J. Mol. Sci. 2016, 17, 1419.

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