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.

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.

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.

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.

Oral immunization with recombinant protein antigen expressed in tobacco against fish nervous necrosis virus.

Nervous necrosis virus (NNV), also known as betanodavirus, has been recently implicated in mass mortalities of cultured marine fish. An effective vaccine is urgently needed to protect fish against this virus. However, parenteral immunization methods are very stressful. Individual immunization for thousands of fish is very labor intensive and expensive. Therefore, we expressed NNV coat protein in tobacco chloroplasts and used it as an oral vaccine to induce immunities in fish followed by challenges with NNV. Our results revealed that mice (IgG and IgA) and fish (IgM) immunized with the oral vaccine developed significantly higher antibody titers against the NNV coat protein. Fish were partially protected against viral challenge. Taken together, our results demonstrated that a plant-based vaccine could effectively induce immune response and protect groupers against NNV. The present method could be used to develop oral fish vaccine in the future.

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.

Potent In Vitro and In Vivo Activity of Plantibody Specific for Porphyromonas gingivalis FimA.

Fimbrial protein fimbrillin (FimA), a major structural subunit of Porphyromonas gingivalis, has been suggested as a vaccine candidate to control P. gingivalis-induced periodontal disease. Previously, cDNAs encoding IgG monoclonal antibodies (MAbs) against purified FimA from P. gingivalis 2561 have been cloned, and the MAbs have been produced in rice cell suspension. Here we examined the biological activities of the plant-produced MAb specific for FimA (anti-FimA plantibody) of P. gingivalis in vitro and in vivo. The anti-FimA plantibody recognized oligomeric/polymeric forms of native FimA in immunoblot analysis and showed high affinity for native FimA (KD = 0.11 nM). Binding of P. gingivalis (10(8) cells) to 2 mg of saliva-coated hydroxyapatite beads was reduced by 53.8% in the presence of 1 µg/ml plantibody. Anti-FimA plantibody (10 µg/ml) reduced invasion of periodontal ligament cells by P. gingivalis (multiplicity of infection, 100) by 68.3%. Intracellular killing of P. gingivalis opsonized with the anti-FimA plantibody by mouse macrophages was significantly increased (77.1%) compared to killing of bacterial cells with irrelevant IgG (36.7%). In a mouse subcutaneous chamber model, the number of recoverable P. gingivalis cells from the chamber fluid was significantly reduced when the numbers of bacterial cells opsonized with anti-FimA plantibody were compared with the numbers of bacterial cells with irrelevant IgG, 66.7% and 37.1%, respectively. These in vitro and in vivo effects of anti-FimA plantibody were comparable to those of the parental MAb. Further studies with P. gingivalis strains with different types of fimbriae are needed to investigate the usefulness of anti-FimA plantibody for passive immunization to control P. gingivalis-induced periodontal disease.

Immunogenicity of an S1D epitope from porcine epidemic diarrhea virus and cholera toxin B subunit fusion protein transiently expressed in infiltrated Nicotiana benthamiana leaves.

Porcine epidemic diarrhea virus (PEDV) belongs to the Coronaviridae family and causes acute enteritis in pigs. A fragment of the large spike glycoprotein, termed the S1D epitope (aa 636-789), alone and fused with cholera toxin B subunit, were independently cloned into plant expression vectors, yielding plasmids pMYV717 and pMYV719, respectively. Plant expression vectors were transformed into Agrobacterium tumefaciens and subsequently infiltrated into Nicotiana benthamiana leaves. The highest expression level of S1D was found at 2 days post infiltration (dpi), reached 0.04 % of total soluble protein, and rapidly decreased thereafter. The expression and assembly of CTB-S1D fusion protein were confirmed by Western blot and GM1-ELISA. The highest expression level of CTB-S1D fusion protein was 0.07 % of TSP at 4 dpi, with a rapid decrease thereafter. In the presence of p19 protein from tomato bushy stunt virus, the S1D and CTB-S1D protein levels peaked at 6 dpi and were fourfold to sevenfold higher than in the absence of p19, respectively. After oral administration of transiently expressed CTB-S1D fusion protein, or with bacterial cholera toxin or rice callus expressing mutant cholera toxin 61F, mice exhibited significantly greater serum IgG and sIgA levels against bacterial CTB and S1D antigen, peaking at week 6. Transiently expressed CTB-S1D fusion protein will be administered orally to pigs to assess the immune response against PEDV.

Dengue virus E glycoprotein production in transgenic rice callus.

Dengue is a disease caused by dengue virus and represents the most important arthropod-borne viral disease in humans. Dengue virus enters host cells via binding of envelope glycoprotein (E) to a receptor. In this study, plant expression vectors containing native and synthetic glycoprotein E genes (sE) modified based on plant-optimized codon usage and fused with an ER retention signal were constructed under control of the rice amylase 3D promoter expression system. Plant expression vectors were introduced into rice callus (Oryza sativa L. cv. Dongin) via particle bombardment-mediated transformation. The integration and expression of target genes were confirmed in the transgenic callus by genomic DNA PCR and Northern blot analyses, respectively. The plant-codon optimized sE gene with an ER retention signal showed high protein production levels based on Western blot analysis of approximately 18.5 ug/g dried calli weight by immunoblot-based densitometric analysis. These results suggest that the plant-codon optimized sE gene with an ER retention signal was highly produced in the transgenic rice callus.

Production of functional human vascular endothelial growth factor(165) in transgenic rice cell suspension cultures.

Vascular endothelial growth factors (VEGFs) are secreted by tumor cells and other cells exposed to hypoxia, and play a critical role in the development and differentiation of the vascular system. In this study, we investigated the production of functional recombinant human VEGF165 (rhVEGF165) in transgenic rice cell suspension culture. Complementary DNA was synthesized from human leukemia HL60 cells and cloned into expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter. The rice seed (Oryza sativa L. cv. Dongjin) was transformed with this recombinant vector by the Agrobacterium mediated method and the integration of the target gene into the plant genome was confirmed by genomic PCR. The expression of rhVEGF165 in the rice cells was determined by Northern blot and Western blot analyses. The accumulated rhVEGF165 protein in the culture medium was 19 mg/L after 18 days of culturing in a sugar-free medium. The rhVEGF165 was purified using a heparin HP column and its biological activity was tested on human umbilical vein endothelial cells (HUVECs). The purified rhVEGF165 significantly increased the proliferative activity of the HUVECs. Therefore, it was demonstrated that functional rhVEGF165 could be produced using transgenic rice suspension culture vector under the control of the RAmy3D promoter.

M cell-targeting ligand and consensus dengue virus envelope protein domain III fusion protein production in transgenic rice calli.

The spread of dengue (DEN) virus is becoming a major concern due to the possibility of primary infection with one of the four dengue serotypes (DEN 1-4) and secondary infection with other heterotypes, which can further aggravate clinical manifestations. A gene encoding consensus envelope protein domain III (cEDIII) of dengue virus with neutralizing activity against four dengue virus serotypes was fused to M cell-targeting peptide ligand (Co1) to increase its mucosal immunogenicity and was introduced into rice calli under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of scEDIII-Co1 fusion gene in transgenic rice calli were confirmed by genomic DNA PCR amplification, Northern and Western blot analyses, respectively. The deliveries of cEDIII-Co1 fusion proteins into mucosal immune inductive site (including M cells) were confirmed by in vitro and in vivo antigen uptake assays. These results showed that plant-produced M cell-targeting peptide ligand, Co1, fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of immune responses.

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.

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.

Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.).

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB-sCOE), and the sCTB-sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB-sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB-COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB-COE fusion protein to its receptor, G(M1)-ganglioside, in transgenic plants was confirmed via G(M1)-ELISA with anti-cholera toxin and anti-COE antibody. Based on G(M1)-ELISA, the expression level of CTB-COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB-COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.

(E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea inhibits proliferation of MCF-7 cells through G1 cell cycle arrest and mitochondria-mediated apoptosis.

Growing interest in the beneficial effects of antioxidants has inspired the synthesis of new phenolic acid phenethyl ureas (PAPUs) with enhanced antioxidant potential. We have previously shown the capacity of one PAPU compound, (E)-1-(3,4-dihydroxyphenethyl)-3-styrylurea (PAPU1), to induce caspase-dependent apoptosis in melanoma cells. In the present study, we examined the anti-proliferative effects of PAPU compounds on MCF-7 human breast cancer cells and determined the molecular mechanisms involved. Treatment with PAPU compounds inhibited predominantly proliferation in these cells, where the PAPU1 was the most efficient form. Flow cytometric analysis showed that PAPU1 blocked cell cycle progression in the G(0)/G(1) phase, and reduced the proportion of cells in G(2)/M phase. This was related to the inhibition of cell cycle regulatory factors, including cyclin D/E and cyclin-dependent kinase (CDK) 2/4, through induction of p21(Cip1). PAPU1 also induced the mitochondrial-mediated and caspase-dependent apoptosis in MCF-7 cells. This was evidenced by cellular changes in the levels of Bcl-2 and Bax, loss of the mitochondrial membrane potential, release of cytochrome c into the cytosol, and caspase-9 activation. Collectively, our results suggest that G(1) cell cycle regulatory proteins and mitochondrial pathways are the crucial targets of PAPU1 in the chemoprevention of breast cancer cells.

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.

Over-expression of JunB inhibits mitochondrial stress and cytotoxicity in human lymphoma cells exposed to chronic oxidative stress.

Activator protein-1 can induce either cell survival or death, which is controlled by opposing effects of different Jun members. It is generally accepted that c-Jun is pro-apoptotic, but that JunD is anti-apoptotic in stress-exposed cells. Additionally, although there are reports suggesting that JunB plays a protective role, its role in stress-induced apoptosis remains unclear. Here, we investigated the role of JunB in H(2)O(2)-induced cell death using cells that over-expressed the protein or were transfected with si-JunB. Inhibition of JunB expression accelerated H(2)O(2)-mediated loss of mitochondrial membrane potential (MMP) and cytotoxicity. Conversely, over-expression of JunB protein led to significant inhibition of the MMP loss and cell death. The increase in JunB expression also attenuated nuclear relocation of apoptosis-inducing factor and mitochondrial Bcl-2 reduction that occurred following H(2)O(2) exposure. These results suggest that JunB can signal survival against oxidant-mediated cell death by suppressing mitochondrial stress. [BMB reports 2010; 43(1): 57-61].

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 immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.