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.
authors.affiliation: Research Center of Bioactive Materials, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors.forename: Ngoc-Luong
authors.initials: NL
authors.lastname: Nguyen
authors.affiliation: Research Center of Bioactive Materials, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors.forename: Kum-Kang
authors.initials: KK
authors.lastname: So
authors.affiliation: Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk 570-749, Republic of Korea.
authors.forename: Jung-Mi
authors.initials: JM
authors.lastname: Kim
authors.affiliation: Research Center of Bioactive Materials, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors.forename: Sae-Hae
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Research Center of Bioactive Materials, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors.forename: Yong-Suk
authors.initials: YS
authors.lastname: Jang
authors.affiliation: Research Center of Bioactive Materials, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Yang
authors.affiliation: Research Center of Bioactive Materials, Center for Fungal Pathogenesis, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea. Electronic address: dhkim@jbnu.ac.kr.
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kim
mesh_terms: D000284:Administration, Oral; D000956:Antigens, Viral / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D003716:Dengue Virus / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D005822:Genetic Vectors / Q000235:genetics; D018928:Immunity, Mucosal / Q000276:immunology; D008024:Ligands; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010581:Peyer's Patches / Q000166:cytology* / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic; D014765:Viral Vaccines / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D000939:Epitopes; D008024:Ligands; D011993:Recombinant Fusion Proteins; D014765:Viral Vaccines
keywords: Baker's yeast; Dengue virus; Domain III of the dengue envelope protein; M cell-specific peptide ligand; Oral feeding