Viral hemorrhagic septicemia virus glycoprotein production in tobacco.

Viral hemorrhagic septicemia virus (VHSV) causes mortality in numerous marine and freshwater fish species resulting in heavy losses in fish farming. The glycoprotein gene of VHSV was fused with the cholera toxin B subunit (CTB) and expressed transiently in leaf tissues of Nicotiana benthamiana via the agroinfiltration method. The glycoprotein gene was divided into two parts to improve assembly of CTB fusion proteins (CTB-VHSV99-235 and CTB-VHSV258-417). Production of CTB fusion proteins was confirmed in the agroinfiltrated leaf tissue by western blot analysis. The plant-produced CTB fusion proteins showed biological activity to GM1-ganglioside, a receptor for biologically active CTB, on GM1-ELISA. The expression level of the CTB-VHSV fusion proteins was 0.86% (CTB-VHSV99-235) and 0.93% (CTB-VHSV258-417) of total proteins in agroinfiltrated leaf tissue, as determined by GM1-ELISA. These results suggest that Agrobacterium-mediated transient expression of CTB fusion antigens of VHSV is a rapid and convenient method and demonstrate the feasibility of using agroinfiltrated plant leaf tissues expressing CTB-fusion antigens as a plant-based vaccine to prevent VHSV infection.

Production and immunogenicity of Actinobacillus pleuropneumoniae ApxIIA protein in transgenic rice callus.

Actinobacillus pleuropneumoniae is a major etiological agent that is responsible for swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA is one of the virulence factors in A. pleuropneumoniae and has been considered as a candidate for developing a vaccine against the bacterial infection. A gene encoding an ApxIIA fragment (amino acids 439-801) was modified based on a plant-optimized codon and constructed into a plant expression vector under the control of a promoter and the 3' UTR of the rice amylase 3D gene. The plant expression vector was introduced into rice embryogenic callus (Oryza sativa L. cv. Dongjin) via particle bombardment-mediated transformation. The integration and transcription of the ApxIIA439-801 gene were confirmed by using genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis of ApxIIA439-801 antigen protein in transgenic rice callus was confirmed by western blot analysis. The concentration of antigen protein in lyophilized samples of transgenic rice callus was 250 µg/g. Immunizing mice with protein extracts from transgenic plants intranasally elicited secretory IgA. These results demonstrate the feasibility of using a transgenic plant to elicit immune responses against A. pleuropneumoniae.

Efficient Syntheses of 1,2,3-Triazoloamide Derivatives Using Solid- and Solution-Phase Synthetic Approaches.

Efficient synthetic routes for the preparation of secondary and tertiary 1,2,3-triazoloamide derivatives were developed. A secondary α-1,2,3-triazoloamide library was constructed and expanded by a previously developed solid-phase synthetic route and a tertiary 1,2,3-triazoloamide library was constructed by a parallel solution-phase synthetic route. The synthetic routes rely on amide formation with secondary amines and chloro-acid chlorides; SN2 reaction with sodium azide; and the selective [3 + 2] Hüisgen cycloaddition with appropriate terminal alkynes. The target secondary and tertiary 1,2,3-triazoloamide derivatives were obtained with three-diversity points in excellent overall yields and purities using the reported solid- and solution-phase synthetic routes, respectively.

Fabrication of Tungsten Oxide Nanowalls through HFCVD for Improved Electrochemical Detection of Methylamine.

In this study, well-defined tungsten oxide (WO3) nanowall (NW) thin films were synthesized via a controlled hot filament chemical vapor deposition (HFCVD) technique and applied for electrochemical detection of methylamine toxic substances. Herein, for the thin-film growth by HFCVD, the temperature of tungsten (W) wire was held constant at ~1450 °C and gasification was performed by heating of W wire using varied substrate temperatures ranging from 350 °C to 450 °C. At an optimized growth temperature of 400 °C, well-defined and extremely dense WO3 nanowall-like structures were developed on a Si substrate. Structural, crystallographic, and compositional characterizations confirmed that the deposited WO3 thin films possessed monoclinic crystal structures of high crystal quality. For electrochemical sensing applications, WO3 NW thin film was used as an electrode, and cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were measured with a wide concentration range of 20 µM~1 mM of methylamine. The fabricated electrochemical sensor achieved a sensitivity of ~183.65 µA mM-1 cm-2, a limit of detection (LOD) of ~20 µM and a quick response time of 10 s. Thus, the fabricated electrochemical sensor exhibited promising detection of methylamine with considerable stability and reproducibility.

Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli.

To increase immune responses of plant-based vaccines in intestine mucosal immune systems, a synthetic neutralizing epitope (sCOE) gene of porcine epidemic diarrhea virus (PEDV) was fused with M cell-targeting ligand (Co1) and introduced into a plant expression vector under the control of rice amylase 3D promoter. The sCOE-Co1 fusion gene was introduced into rice calli via the particle bombardment-mediated transformation method. The stable integration and transcriptional expression of the sCOE-Co1 fusion gene was confirmed by genomic DNA PCR amplification and Northern blot analysis, respectively. The expression of the COE-Co1 fusion protein was confirmed by immunoblot analysis. The highest expression level of the COE-Co1 fusion protein reached 0.083 % of the total soluble protein according to quantitative densitometry of Western blot analysis. Mice immunized with transgenic rice calli protein extracts induced significant serum IgG and fecal IgA antibody levels against purified bacterial COE. The systemic and mucosal immune responses were confirmed by measuring COE-specific IgG and IgA antibody-secreting cells in the lymphocytes extracted from the spleen and COE-specific IgA antibody-secreting cells in the Peyer's patches from immunized mice. These results indicated that oral immunization of plant-produced COE-Co1 fusion protein could elicit efficient systemic and mucosal immune responses against the COE antigen. Key message Neutralizing epitope from porcine epidemic diarrhea virus-M cell targeting ligand fusion protein was produced in transgenic rice calli and elicited systemic and mucosal immune responses by oral administration in mice.

Astaxanthin Protects against Hyperglycemia-Induced Oxidative and Inflammatory Damage to Bone Marrow and to Bone Marrow-Retained Stem Cells and Restores Normal Hematopoiesis in Streptozotocin-Induced Diabetic Mice.

Hyperglycemia has various adverse health effects, some of which are due to chronic oxidative and inflammatory impairment of bone marrow (BM), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). Astaxanthin (ASTX) has been shown to ameliorate hyperglycemia-associated systemic complications and acute mortality, and this effect is partially associated with restoration of normal hematopoiesis. Here, the effects of ASTX on diabetes-induced complications in BM and BM stem cells were investigated, and the underlying molecular mechanisms were elucidated. Ten-week-old C57BL/6 mice received a single intraperitoneal injection of streptozotocin (STZ; 150 mg/kg) in combination with oral gavage of ASTX (12.5 mg/kg) for 30 or 60 consecutive days. Supplemental ASTX ameliorated acute mortality and restored the STZ-impaired bone mass accrual and BM microenvironment in STZ-injected mice. Oral gavage of ASTX suppressed osteoclast formation in the BM of STZ-injected mice. Specifically, supplementation with ASTX inhibited oxidative stress and senescence induction of BM HSCs and MSCs and ameliorated hematopoietic disorders in STZ-injected mice. These effects of ASTX were associated with BM restoration of angiopoietin 1, stromal cell-derived factor 1, ß-catenin, and Nrf2. Long-term ASTX gavage also recovered the STZ-induced dysfunction in migration, colony formation, and mineralization of BM-derived stromal cells. Further, a direct addition of ASTX exhibited direct and dose-dependent inhibition of osteoclastic activation without cytotoxic effects. Collectively, these results indicate that ASTX protects against diabetes-induced damage in the BM microenvironment in BM, HSCs, and MSCs and restores normal hematopoiesis and bone accrual in STZ-injected mice.

Cloning and characterization of heavy and light chain genes encoding the FimA-specific monoclonal antibodies that inhibit Porphyromonas gingivalis adhesion.

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis.

Cholera toxin B subunit-domain III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.

Envelope glycoprotein E of the dengue virus, which plays a crucial role in its entry into host cells, has an immunogenic domain III (EIII, amino acids 297-394), which is capable of inducing neutralizing antibodies. However, mice immunized with EIII protein without adjuvant elicited low immune responses. To improve low immune responses, a DNA fragment, consisting of cholera toxin B subunit and EIII gene (CTB-EIII), was constructed and introduced into tobacco plant cells (Nicotiana tabacum L. cv. MD609) by Agrobacterium tumefaciens-mediated transformation methods. The integration and transcription of CTB-EIII fusion gene were confirmed in transgenic plants by genomic DNA PCR amplification and Northern blot analysis, respectively. The results of immunoblot analysis with anti-CTB and anti-dengue virus antibodies showed the expression of the CTB-EIII fusion protein in transgenic plant extracts. Based on the G(M1)-ELISA results, the CTB-EIII protein expressed in plants showed the biological activity for intestinal epithelial cell membrane glycolipid receptor, G(M1)-ganglioside, and its expression level was up to about 0.019% of total soluble protein in transgenic plant leaf tissues. The feasibility of using a plant-produced CTB-EIII fusion protein to generate immunogenicity against domain III will be tested in future animal experiments.

Tissue culture and expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic Peperomia pellucida.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a non-toxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. We synthesized a gene encoding the LTB adapted to the optimized coding sequences in plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its expression level and protein assembly in plants. The synthetic LTB gene was located into a plant expression vector under the control of CaMV 35S promoter and was introduced into Peperomia pellucida by biolistic transformation method. The integration of synthetic LTB gene into genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification method. The assembly of plant-produced LTB was detected by western blot analysis. The amount of LTB protein produced in transgenic P. pellucida leaves was approximately 0.75% of the total soluble plant protein. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is receptor for LTB on the cell surface, suggesting that the LTB subunits formed biological active pentamers.

Expression and assembly of cholera toxin B subunit (CTB) in transgenic carrot (Daucus carota L.).

We expressed the cholera toxin B subunit (CTB) fused to an endoplasmic reticulum retention signal (SEKDEL) in carrot roots using an Agrobacterium-mediated transformation method. Fourteen independent transgenic lines were regenerated via somatic embryogenesis after 6 months of culture. The sCTB gene was detected in the genomic DNA of transgenic carrot by PCR amplification. Expressions and assembly of sCTB protein into oligomeric structures of pentameric size were observed in transgenic plant extracts by Western blot analysis. The sCTB produced by transgenic carrot roots demonstrated strong affinity for GM1-ganglioside, suggesting that the sCTB conserved the antigenic sites for binding and proper folding of the pentameric sCTB structure. The expression level of sCTB comprised approximately 0.48% of total soluble protein (TSP) in root of transgenic carrot.

Immunogenicity of a cholera toxin B subunit Porphyromonas gingivalis fimbrial antigen fusion protein expressed in E. coli.

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.

Expression of functional pentameric heat-labile enterotoxin B subunit of Escherichia coli in Saccharomyces cerevisiae.

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmic reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active oligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

Production of a heat-labile enterotoxin B subunit-porcine epidemic diarrhea virus-neutralizing epitope fusion protein in transgenic lettuce (Lactuca sativa).

Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus (sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026∼0.048% of the total soluble protein in the transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization.

Ethanol extract of Dryopteris crassirhizoma alleviates allergic inflammation via inhibition of Th2 response and mast cell activation in a murine model of allergic rhinitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Dryopteris crassirhizoma (DC) is used as a traditional herbal remedy to treat various diseases, the tapeworm infection, common cold, and cancer in Korea, Japan, and China. DC also has the antioxidant anti-inflammatory and antibacterial activities. However, the anti-allergic inflammatory effect of DC and some of its mechanisms in allergic rhinitis model are unknown well. AIM OF THIS STUDY: The purpose of this study is to investigate the anti-allergic inflammatory effect of DC on the allergic rhinitis model, mast cell activation and histamine release. MATERIALS AND METHODS: Allergic rhinitis was induced in BALB/c mice by sensitization and challenge with ovalbumin (OVA). Different concentration of DC and dexamethasone was administrated by oral gavage on 1 h before the OVA challenge. Mice of the control group were treated with saline only. Then mice were evaluated for the presence of nasal mucosa inflammation, the production of allergen-specific cytokine response and the histology of nasal mucosa. RESULTS: DC significantly ameliorated the nasal symptoms and the inflammation of nasal mucosa. DC also reduced the infiltration of eosinophils and mast cells in these tissues and the release of histamine in blood. Meanwhile, DC evidently inhibited the overproduction of Th2 cytokines and increased the Th1 and Treg cytokines in nasal lavage fluid by OVA. DC also reduced the levels of OVA-specific IgE, IgG1 and IgG2a in blood. CONCLUSIONS: This study suggests that DC has a significant anti-allergic inflammatory effect in the nasal cavity. DC may have the therapeutic effect of allergic rhinitis.

Co-expression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture.

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.

Expression of cholera toxin B subunit in transgenic rice endosperm.

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Isolation and characterization of antioxidation enzymes from cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 5-l bioreactor.

In this study, a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe) was developed, using 50 g/l of fresh weight inoculum in a batch culture. The highest cell biomass obtained from a 5-l bioreactor equipped with three impellers after 14 days of culture was utilized to extract secondary metabolites (essential oil and curcumin) and determine the activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase). For essential oil and curcumin, zedoary extracts were recovered via a variety of methods: steam distillation, volatile solvents, and Soxhlet. After 14 days of culture using volatile solvents, the optimal yield of essential oil (1.78%) was obtained when using petroleum ether at 40 degrees C in 6 h of extraction, and the best curcumin yield (9.69%) was obtained at 60 degrees C in 6 h via extraction with 90% ethanol. The activities of antioxidant enzymes from zedoary cells were also assessed. The specific activities of peroxidase, superoxide-dismutase, and catalase reached maximum values of 0.63 U/mg of protein, 16.60 U/mg of protein, and 19.59 U/mg of protein after 14 days of culture, respectively.

Effect of phlorotannins on myofibroblast differentiation and ECM protein expression in transforming growth factor ß1­induced nasal polyp­derived fibroblasts.

Phlorotannins (PTNs), a group of phenolic compounds from seaweeds, have diverse bioactivities. However, there has been no report on their antifibrotic effects during nasal polyp (NP) formation. In the present study, the effect of PTNs on transforming growth factor (TGF)­ß1­induced profibrotic responses in nasal polyp­derived fibroblasts (NPDFs) were determined and the relevant signaling pathways were investigated. The expression levels of collagen type­1 (Col­1) and fibronectin in NP tissues were measured by western blot analysis and immunohistochemistry. The NPDFs were treated with TGF­ß1 (1 ng/ml) in the presence or absence of PTNs (5­30 µg/ml). The expression levels of α­smooth muscle actin (α­SMA), Col­1, fibronectin, and phosphorylated­small mothers against decapentaplegic (Smad)2/3 in NPDFs were measured by western blot analysis. The contractile activity of the NPDFs was determined by a collagen gel contraction assay. Col­1 and fibronectin proteins were found to be expressed in NP tissues. PTNs had no significant cytotoxic effect on TGF­ß1­induced NPDFs. TGF­ß1 induced the expression α­SMA, Col­1 and fibronectin, and stimulated fibroblast­mediated contraction of collagen gel. However, pre­treatment with PTNs inhibited the expression of these proteins. The inhibitory effects were mediated through the suppression of Smad2/3 signaling pathways in TGF­ß1­induced NPDFs. These resulted suggested that PTNs may be important in inhibiting myofibroblast differentiation and extracellular matrix protein accumulation in NP formation through the Smad2/3 signaling pathway.

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Surface displayed expression of a neutralizing epitope of spike protein from a Korean strain of porcine epidemic diarrhea virus.

The neutralizing epitope (K-COE) of the spike protein from a Korean strain of porcine epidemic diarrhea virus (PEDV) has been shown to prevent and foster an immune response to PED, when orally adjusted. The cell surface of the budding yeast,Saccharomyces cerevisiae, was engineered to anchor the K-COE on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for animal feed, with immunogenic functions. In this study, the K-COE gene (K-COE) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (Ramy 1A), was fused with the gene encoding the carboxyterminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a mating associated protein that is anchored covalently to the cell wall. The glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was selected in order to direct the expression of the fusion construct, and the resulting recombinant plasmid was then introduced intoS. cerevisiae. The surface display of K-COE was visualized via confocal microscopy using a polyclonal antibody against K-COE as the primary antibody, and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG as the secondary antibody. The display of the K-COE on the cell surface was further verified via Western blot analysis using the cell wall fraction after the administration of α-1,3-glucanase/PNGase F/ß-mannosidase treatment.