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Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae

Paper ID Volume ID Publish Year Pages File Format Full-Text
20301 43168 2015 9 PDF Available
Title
Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae
Abstract

A fusion construct (Tet-EDIII-Co1) consisting of an M cell-specific peptide ligand (Co1) at the C-terminus of a recombinant tetravalent gene encoding the amino acid sequences of dengue envelope domain III (Tet-EDIII) from four serotypes was expressed and tested for binding activity to the mucosal immune inductive site M cells for the development of an oral vaccine. The yeast episomal expression vector, pYEGPD-TER, which was designed to direct gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator, was used to clone the Tet-EDIII-Co1 gene and resultant plasmids were then used to transform Saccharomyces cerevisiae. PCR and back-transformation into Escherichia coli confirmed the presence of the Tet-EDIII-Co1 gene-containing plasmid in transformants. Northern blot analysis of transformed S. cerevisiae identified the presence of the Tet-EDIII-Co1-specific transcript. Western blot analysis indicated that the produced Tet-EDIII-Co1 protein with the expected molecular weight was successfully secreted into the culture medium. Quantitative Western blot analysis and ELISA revealed that the recombinant Tet-EDIII-Co1 protein comprised approximately 0.1–0.2% of cell-free extracts (CFEs). In addition, 0.1–0.2 mg of Tet-EDIII-Co1 protein per liter of culture filtrate was detected on day 1, and this quantity peaked on day 3 after cultivation. In vivo binding assays showed that the Tet-EDIII-Co1 protein was delivered specifically to M cells in Peyer's patches (PPs) while the Tet-EDIII protein lacking the Co1 ligand did not, which demonstrated the efficient targeting of this antigenic protein through the mucosal-specific ligand.

Keywords
Oral feeding; M cell-specific peptide ligand; Dengue virus; Domain III of the dengue envelope protein; Baker's yeast
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Expression and characterization of an M cell-specific ligand-fused dengue virus tetravalent epitope using Saccharomyces cerevisiae
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 119, Issue 1, January 2015, Pages 19–27
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us