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Expression of the Escherichia coli heat-labile enterotoxin B subunit in transgenic watercress (Nasturtium officinale L.)
Abstract
A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB) gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein. GM1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized LTB subunits formed biologically active pentamers.
... Transgenic watercresses (Nasturtium officinale L.) developed with the construct of pMYO51 vector for LTB expression (Kang et al. 2004). Four plants (#1, #3, #4 and #5 were renamed A1, A3, A4 and A5) were transferred the synthetic LTB gene through Agrobacterium tumefaciens -mediated transformation (Loc et al. 2011), the others (#1 and #4 were renamed B1 and B4) by using biolistic method (Loc et al. 2010b). Transgenic plants were maintained in in vitro condition on the MS (Murashige and Skoog 1962) medium supplemented with 3 % sucrose, 0.8 % agar and 1 mg l −1 napthaleneacetic acid. ...
... Aliquots (10 μg) of total genomic DNA from the leaves of the transgenic plant and the wild-type were digested with KpnI and electrophoresed on a 0.8 % agarose gel, which was then transferred to Hybond-N+ membrane (Amersham). The blot was hybridized with a probe (synthetic LTB gene, 414 bp) labeled with digoxigenin- (Loc et al. 2010b(Loc et al. , 2011, except optical density was measured at 490 nm. Total soluble protein content is determined by the method of Bradford (1976) using bovine serum albumin as a standard. ...
... In this work, the pentamer structure of the plant-derived LTB protein had slightly higher molecular weight than that of the bacterial LTB protein (45 kDa). However, our previous studies in tobacco (Kang et al. 2003), tomato (Nhi et al. 2010), Peperomia pellucida (Loc et al. 2010a), even in in vitro watercress (Loc et al. 2010b and2011) indicated that plantderived LTB protein had molecular weight of 45 kDa equal that of bacteria. This discrepancy was presumably due to the extra six amino acids that were added at the C-terminus for endoplasmic reticulum retention (Kang et al. 2004), and the failure of the plant cells to remove the leader signal peptide (Arakawa et al. 1997). ...
In this work, the characterizations of the LTB (Escherichia coli heat-labile toxin B subunit) transgenic watercresses through Agrobacterium tumefaciens-mediated transformation (A1 and A3-A5) and by using biolistic method (B1 and B4) were investigated. Generally, their growth is not remarkably different from the wild-type. Their physiological and biochemical characteristics are relatively different in which plant A1 has highest values such as pigment (0.92 mg g-1), photosynthetic rate (23.39 mgCO2 [dm2]-1 h-1), dry matter (7.42%), vitamin C (0.34 mg g-1), calcium (0.83 mg g-1), and potassium (2.47 mg g-1). The dry matter and calcium of all the transgenic watercresses and the wild-type are the same content. Southern blot hybridization showed the transgenic plants contain 1-2 copies in the genome. LTB protein strongly expresses in all the transgenic plants with contents from 1.16 to 1.46% of total soluble protein. The GM1-ELISA binding assay indicated that plant-derived LTB protein bound to GM1-ganglioside receptors.
... These alternative subunit vaccines have several advantages, such as low-cost of production and friendly administration (Mason et al. 2002). Several plant models, such as tobacco, potato, maize, ginseng, carrot, and watercress have been utilized to assess the feasibility of developing a plant-based vaccine against ETEC (Haq et al. 1995; Mason et al. 1998; Chikwamba et al. 2002; Kang et al. 2006; Rosales-Mendoza et al. 2007; Loc et al. 2011 ). In general, these studies have confirmed that plantderived LTB retains both its antigenicity and immunogenicity compared with the native LTB. ...
... As LTB has immunoprotective and adjuvant properties, it has been produced in several plants in an effort to assess the feasibility of oral immunization using a low-cost source of the recombinant protein. Different food (potato, rice, soybean, maize, carrot, and watercress) and non-food (tobacco, ginseng) crops have been used as models for the production of LTB (Haq et al. 1995; Mason et al. 1998; Moravec et al. 2007; Chikwamba et al. 2002; Kang et al. 2006; Rosales-Mendoza et al. 2007; Kim et al. 2010; Loc et al. 2011). Kim et al. (2007) have reported on the synthesis and assembly of the LTB protein into oligomeric structures of pentameric size in lettuce, reaching expression levels of up to 1.0–2.0% of the total soluble protein. ...
... Kim et al. (2007) have reported on the synthesis and assembly of the LTB protein into oligomeric structures of pentameric size in lettuce, reaching expression levels of up to 1.0–2.0% of the total soluble protein. Recently, Loc et al. (2011) reported on the expression of an optimized LTB gene codon in watercress. Western blot and GM1-ELISA analyses revealed that the watercressderived LTB protein was antigenic and assembled into a pentameric form. ...
Transgenic plants serve as attractive systems for the production and delivery of subunit vaccines, thus expression of an enterotoxigenic
Escherichia coli (ETEC) antigen in an edible plant may lead to the development of a viable oral vaccine against cholera and ETEC diarrhea.
In this study, expression of the heat labile toxin B subunit (LTB) from ETEC was performed in lettuce, and its immunological
characterization was investigated. A total of 27 independent transgenic lines were established following Agrobacterium-mediated transformation. Selected lettuce lines were subjected to GM1-ELISA to confirm the proper quaternary structure of
the LTB protein. Levels of accumulation of the pentameric LTB reached up to 0.05% of the total soluble protein (TSP) in T1
and T2 progenies of these lines. Oral immunization of Balb/c mice was conducted using three weekly doses of lettuce-derived
LTB. This elicited specific and significant antibody responses in both serum and intestinal tissues. Moreover, mice immunized
with lettuce-derived LTB showed diminished intestinal fluid accumulation following challenge with the cholera toxin. This
study demonstrated that this plant-based vaccine may contribute to immunization practices against diarrheal diseases.
KeywordsEnteric diseases–Plant-based vaccine–Oral immunization
... Ten-day-old cotyledons were excised from in vitro-germinated seedlings and precultured on the shoot regeneration medium for 2 days. Then, we dropped the Agro-suspension onto the cotyledons (two drops onto each cotyledon) with a pipette (Loc et al. 2011). After 2 days of cocultivation, cotyledons were cultured for 10 days on selection medium with 35 mg/l kanamycin (Km) and 200 mg/l cefotaxime (Cef ), for 10 days on selection medium with 50 mg/l Km and 200 mg/l Cef, and for 4 weeks on selection medium with 100 mg/l Km and 200 mg/l Cef. ...
... Western blot analysis of these fruits detected a pentameric LTB protein with a molecular weight of about 45 kDa (Figure 5A). This result is similar to those obtained from different plant expression systems, such as those of tobacco (Kang et al. 2003), lettuce (Kim et al. 2007), Peperomia pellucida (Loc et al. 2010a), and watercress (Loc et al. 2011). However, there were only fruits of two transgenic plants expressing the LTB protein (#1 and #3). ...
We report a feasibility study for expressing the LTB protein (Escherichia coli heat-labile enterotoxin B subunit)
via Agrobacterium-mediated transformation of tomato (Solanum lycopersicum L.). We produced five regenerated plants obtained on the selection medium supplemented with an antibiotic. Stable integrations of the LTB gene into the genome of these plants were confirmed by Southern blot hybridization. Western blot analysis showed that only two of the five T0 transgenic tomato plants expressed the pentameric LTB protein in the fruits. An enzyme-linked immunosorbent assay indicated that these two plants synthesized the LTB protein bound specifically to GM1 ganglioside, suggesting that the LTB subunits formed active pentamers. The LTB protein produced in tomatoes can be a potential candidate for inexpensive, safe, and effective plant-based vaccines.
... Nevertheless, it is possible to identify individuals with higher levels of expression. For example, Loc et al. (2011) used a gene encoding the B subunit of E. coli heat labile enterotoxin (LTB) adapted to plant expression and fused to the endoplasmatic reticulum retention signal SEKDEL to transform watercress (Nasturtium officinalis). Among transformants, it was possible to identify high levels of production of the LTB protein up to 1.3% of total protein were produced, forming biologically active pentamers. ...
The concept of plant biotechnology has radically evolved since its first applications, mainly focused on cell cultures as sources of products used for humans. The advent of recombinant DNA technology as well as new advances in genomics, proteomics or metabolomics has changed the scenario. Modern plant biotechnology incorporates all technology available with different objectives. Firstly, in vitro culture has experienced great advances, contributing to different applications aimed at the development of new plant varieties. Moreover, improvements in genetic engineering have allowed generation of genetically modified plants with very different purposes such as increase plant productivity or produce chemical compounds of medicinal or industrial uses, among others. Also, the advances in molecular biology have made possible to develop high-throughput analyses at the level of genes, proteins, or metabolites.
The objective of this chapter is to briefly summarize recent advances in plant biotechnology related to in vitro culture, genetic engineering and molecular markers and genomics. The increasing speed at which these advances are taking place makes exhaustive review of practical achievements unaffordable. In this sense, the works referred in this chapter just represent specific examples of the advances in each area.
... Transgenic plant systems that express antigens (''edible vaccines'') have several advantages over conventional vaccines such as safety, low production costs, ease of production scale-up, and minimal risk of contamination by animal or human pathogens and toxins. A plant expression system is the most promising edible vaccine system and has the ability to deliver antigens to the mucosal immune systems because the edible part or purified proteins in a transgenic plant expressing antigen proteins can be administrated orally (Lau and Korban 2009;Kim et al. 2006Kim et al. , 2011Loc et al. 2011;Youm et al. 2010;Martinez-Gonzalez et al. 2011). However, a large biomass of transgenic plants containing antigen proteins is required to avoid oral tolerance due to low levels of expression of the antigen and degradation prior to immune priming. ...
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.
... Since the production of recombinant proteins in transgenic plants was first reported in 1986, many such recombinant proteins have been produced in both transgenic plants and plant cell suspension cultures (Hellwig et al. 2004;Obembe et al. 2011;Loc et al. 2011). Plant cell suspension culture systems have been utilized as factories to produce pharmaceutically and industrially useful recombinant proteins due to easy purification and low production cost. ...
A rice cell suspension culture with the rice α-amylase 3D promoter expression system which is induced by sucrose starvation was previously reported to generate a good yield of recombinant proteins. However, this expression system is limited by the accumulation of undesirable α-amylase and proteases in the culture medium. Rice α-amylase is a dominant protein at 43 % of total secreted proteins, and cysteine proteinase (CysP) is a major secreted protease in rice cell suspension cultures following induction via sugar depletion. Here, we nearly eliminated rice α-amylase and CysP proteinase via RNA interference (RNAi) technology to improve the recombinant protein yield in rice cell suspension culture. The effects of RNAi were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis with anti-CysP antibody, and quantitative real-time reverse transcription polymerase chain reaction analysis (RT-PCR). The mRNA levels of α-amylase and CysP were reduced by 94.8 and 95.0 %, respectively. Transgenic rice cell suspension cultures expressing both human granulocyte–macrophage colony-stimulating factor (hGM-CSF) and ihpRNA of α-amylase and CysP genes evidenced a reduction of α-amylase and CysP activity and up to 2.4-fold improvement of hGM-CSF production compared to that in a transgenic cell line expressing hGM-CSF only. Our rice cell suspension culture for the reduction of α-amylase accumulation and protease activity in the culture medium could improve recombinant protein production as an efficient protein expression system using RNA interference technology in plant biotechnology.
... Many vaccine candidates against bacterial and viral pathogens have been produced in transgenic plant expression systems, as this method has the potential to meet the demand for safe and inexpensive vaccines (Loc et al. 2011;Martinez-Gonzalez et al. 2011). However, low immune response and oral immune tolerance, which both are due to the low expression levels of target antigens in transgenic plants, are a barrier to plant-based oral vaccine development. ...
Dengue (DEN) is one of the most important emerging mosquito-borne viral human diseases. Therefore, an effective dengue vaccine with immune responses against all four dengue virus serotypes is highly needed. A fusion gene encoding a synthetic consensus envelope protein domain III (scEDIII) of dengue virus with neutralizing activity against the four dengue virus serotypes and with the B subunit of cholera toxin (CTB) to increase its mucosal immunogenicity was constructed and was introduced into rice callus under the control of the inducible rice amylase 3D promoter expression system. The integration and expression of the CTB-scEDIII fusion gene in transgenic rice callus were confirmed by genomic DNA PCR amplification, Northern, and Western blot analyses, respectively. The biological binding activity of the CTB-cEDIII fusion protein to its GM1-ganglioside receptor was confirmed via GM1-ELISA with anti-CT and anti-dengue virus antibodies. Delivery of the CTB-cEDIII fusion protein into mucosal immune inductive sites (including M cells) in BALB/c mice was confirmed by in vitro and in vivo antigen uptake assays. These results showed that the CTB-cEDIII fusion protein was produced in the transgenic rice callus, and that plant-produced ligand fusion antigen proteins have the potential to be targeted to the mucosal immune system for improvement of the overall immune responses.
Ethnopharmacological relevance:
With a net turnover worth of £181 billion, the cosmetic industry is a leading worldwide business with a very lucrative future. Nonetheless, due to recent concerns regarding toxicity of synthetic cosmetics, herbal products have come into the limelight of cosmetology. The tropical island of Mauritius has a well-anchored diversity of indigenous plant species which are exploited for various purposes but no study has been designed to (i) quantitatively document, (ii) assess the effectiveness, and (iii) study the incidence of adverse effects and perception associated with the use of herbal products for cosmetic applications.
Method:
Data was collected from herbal users via face-to-face interviews using semi-structured questionnaire from key informants. Quantitative ethnobotanical indices (fidelity level (FL), variety of use (VU) and relative frequency of citation (RFC)) were calculated.
Results:
Twenty five herbs belonging to 21 families were recorded in use for 29 different cosmetics applications. Many of the documented species represented well-known plants, although we also recorded a few plants being exploited for new cosmetic applications. Plants with the highest RFC were Curcuma longa L. (0.45), Lawsonia inermis L. (0.42) and Aloe vera (L.) Burm.f. (0.42). A total of 8 plants were reported to score 100% with respect to the FL. Interestingly, Lawsonia inermis L. being the highly cited plant species showed a clear dominance as a popular phytocosmetic and which has also been extensively documented for its pharmacological properties. Moreover, it was found that 25% of the respondents experienced adverse effects; with pruritus (11%) being the most reported condition. It was also observed that participants perceived herbs/herbal products to be free from adverse effects.
Conclusion:
Most of the plants reported have been described in previous studies for their bioactive components which tend to justify their use as phytocosmetics. Further research should be geared to explore the potential of these plant products for the cosmetic industry.
Plant expression system has been firmly attracting researcher's attention in the past two decades and many kinds of plants were employed to express various recombinant proteins. Among those plants, lettuce has been used as a promising expression host for heterologous recombinant proteins due to its advantage of oral delivery without heating process, besides other general superiorities of plant-based expression system. Many transgenic researches on lettuce have been reported and various products have been expressed successfully in lettuce, including vaccine antigens, antibodies, bioactive peptides, therapeutic proteins, industrial products and other interesting proteins. This paper reviews the current literatures on lettuce as expression platform for heterologous proteins, as well as underlying challenges in the future application.
An effective dengue vaccine should elicit immune responses against all four different dengue virus serotypes. This study optimized the codon usage of a gene encoding consensus dengue virus envelope protein domain III (cEDIII) with cross-neutralizing activity against four dengue virus serotypes for plant expression. Then, a plant expression vector was constructed with this gene under the control of the rice amylase 3D promoter (RAmy3D), which is a strong inducible promoter under sugar starvation conditions. The synthetic cEDIII gene was fused with the RAmy3D signal peptide and ER retention signal, SEKDEL, and was introduced into rice callus by particle bombardment-mediated transformation. The integration and expression of cEDIII gene in transgenic rice callus was confirmed by genomic DNA PCR amplification, Northern blot analysis, and western blot analysis, respectively. Densitometric analysis determined that the highest expression level of the cEDIII protein in lyophilized rice callus was approximately 0.45 mg g−1. These results suggest that it is feasible to use transgenic rice callus to produce the consensus dengue virus envelop protein domain III for edible vaccine purposes.
Transgenic plant-derived vaccines comprise a new type of bioreactor that combines plant genetic engineering technology with an organism's immunological response. This combination can be considered as a bioreactor that is produced by introducing foreign genes into plants that elicit special immunogenicity when introduced into animals or human beings. In comparison with traditional vaccines, plant vaccines have some significant advantages, such as low cost, greater safety, and greater effectiveness. In a number of recent studies, antigen-specific proteins have been successfully expressed in various plant tissues and have even been tested in animals and human beings. Therefore, edible vaccines of transgenic plants have a bright future. This review begins with a discussion of the immune mechanism and expression systems for transgenic plant vaccines. Then, current advances in different transgenic plant vaccines will be analyzed, including vaccines against pathogenic viruses, bacteria, and eukaryotic parasites. In view of the low expression levels for antigens in plants, high-level expression strategies of foreign protein in transgenic plants are recommended. Finally, the existing safety problems in transgenic plant vaccines were put forward will be discussed along with a number of appropriate solutions that will hopefully lead to future clinical application of edible plant vaccines.
Actinobacillus pleuropneumoniae is a major etiological agent of swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic
losses in the swine production industry. ApxIIA, one of the virulence factors in A. pleuropneumoniae, is considered a candidate for the development of a vaccine against this bacterial infection. In this study, a fusion gene
consisting of enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit (LTB) and an ApxIIA fragment (amino acids 619–801) (ApxIIA619–801) was fused to the endoplasmic reticulum (ER) retention signal (SEKDEL) and introduced into a plant expression vector under
the control of a ubiquitin promoter. The plant expression vector was transformed into tobacco Nicotiana tabacum L. cv. MD609 using an Agrobacterium-mediated transformation procedure. The integration and transcription of the LTB fusion gene were confirmed by genomic DNA
PCR amplification and Northern blot analysis, respectively. The synthesis and assembly of LTB fusion protein were evidenced
by Western blot analysis and the GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). A quantitative ELISA used to measure the amount of LTB fusion protein produced in the transgenic plant revealed high
levels of the fusion protein, up to 30μgg−1, in lyophilized leaf tissue. These results demonstrate the feasibility of using a transgenic plant to express the LTB-ApxIIA619–801 fusion protein as a first step towards producing plant-based edible vaccines to elicit immune responses against A. pleuropneumoniae via an oral mucosal delivery system.
KeywordsEnterotoxigenic Escherichia coli heat-labile enterotoxin B subunit–
Actinobacillus pleuropneumoniae
–Plant-based edible vaccine–ApxIIA
The dengue virus envelope glycoprotein (DV-E) has been identified as a promising candidate for the development of a subunit
vaccine and to provide an antigen for diagnostic kits. In this study, cell suspension cultures of Nicotiana tabacum and Morinda citrifolia were transformed and evaluated for production of the DV-E serotype 2 protein. The expression cassette consisted of the DV-E
gene along with a signal peptide at its 5′ end. In addition, a KDEL endoplasmic retention sequence was included in a second
construct to evaluate its influence on accumulation levels of the recombinant protein. Expression cassettes were sub-cloned
into a binary vector pCAMBIA 1305.2, and transformation was carried out using Agrobacterium tumefaciens. Transformed cell lines of both N. tabacum and M. citrifolia accumulated DV-E protein, although those carrying the construct containing the KDEL sequence showed higher accumulation levels
than those without. Overall, cell suspension cultures of N. tabacum were more suitable for dengue antigen production than those of M. citrifolia. The highest levels of protein accumulation (0.71±0.06mg DV-E/L) were observed in tobacco cell lines at 5days of culture,
corresponding to 0.3% of total soluble protein. The recombinant protein was reactive with anti-E antibodies.
KeywordsDengue virus–Envelope protein–Plant cell suspension culture–Recombinant protein–Stable expression
Heat-labile enterotoxin B subunit (LTB) of enterotoxigenie Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL] promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.
Adventitious shoot regeneration could be obtained from more than 80% of the calluses initiated from stem explants of watercress
(Rorippa nasturtium-aquaticum) by using an induction medium and a shoot regeneration medium. The induction medium contained 1.15 μM 2,4-dichlorophenoxyacetic
acid and 5 μM thidiazuron; the shoot regeneration medium was composed of 0.5 μM thidiazuron and 2.25 μM 6-benzylaminopurine.
This regeneration procedure was incorporated into an Agrobacterium-mediated transformation procedure for gene transfer into watercress. Factors affecting transformation included preculture,
selection agents, use of tobacco nurse cells, and the length of coculture. A transgenic line of watercress transformed with
a wild-type Bacillus thuringiensis insecticidal gene, cry1Ia3, was not toxic to larvae of the diamondback moth (Plutella xylostella), presumably due to premature polyadenylation of the transcript encoded by this gene in the plant.
The authors have designed and constructed a plant-optimized synthetic gene encoding the Escherichia coli heat-labile enterotoxin B subunit (LT-B), for use in transgenic plants as an edible vaccine against enterotoxigenic E. coli. Expression of the synthetic LT-B gene in potato plants under the control of a constitutive promoter yielded increased accumulation of LT-B in leaves and tubers, as compared to the bacterial LT-B gene. The plant-derived LT-B assembled into native pentameric structures as evidenced by its ability to bind ganglioside. The authors demonstrated immunogenicity by feeding mice the raw tubers and comparing the anti-LT-B serum IgG and faecal IgA to that produced in mice gavaged with bacterial LT-B. Mice were fed three weekly doses of 5 g tuber tissue containing either 20 or 50 μg LT-B, or gavaged weekly with 5 μg of LT-B from recombinant E. coli. One week after the third dose, mice immunized with potato LT-B had higher levels of serum and mucosal anti-LT-B than those gavaged with bacterial LT-B. Mice were challenged by oral administration of 25 μg LT, and protection assessed by comparing the gut/carcass mass ratios. Although none of the mice were completely protected, the higher dose potato vaccine compared favourably with the bacterial vaccine. These findings show that an edible vaccine against E. coli LT-B is feasible.
A synthetic gene of the B-subunit of Escherichia coli heat-labile toxin, optimized for expression in plants, was designed and synthesized. The recombinant viral vector was constructed on the basis of potato virus X containing the LTB gene instead of the removed triple block of transport genes and the coat protein gene, which provides for LTB expression in plants. The vector is introduced into the plant cells during cell infiltration by agrobacteria incorporating a binary vector, the T-DNA region of which contains a cDNA copy of the recombinant viral genome. Under conditions of posttranscriptional gene silencing inhibition, the LTB yield in Nicotiana benthamiana plants is 1-2% of total soluble protein; in this case, LTB synthesized in plants forms pentameric complexes analogous to those found in the native toxin. The designed viral system of LTB transient expression can be used to obtain in plants a vaccine against enteropathogenic Escherichia coli.
Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile
(LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea
to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding
of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETECH10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described
E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame
deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation
in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island.
Transmission of ColE1/pMB1-derived plasmids, such as pBR322, from Escherichia coli donor strains was shown to be an efficient way to introduce these plasmids into Agrobacterium. This was accomplished by using E. coli carrying the helper plasmids pGJ28 and R64drd11 which provide the ColE1 mob functions and tra functions, respectively. For example, the broad host-range replication plasmid, pGV1150, a co-integrate plasmid between pBR322 and the W-type mini-Sa plasmid, pGV1106, was transmitted from E. coli to A. tumefaciens with a transfer frequency of 4.5 x 10(-3). As pBR322 clones containing pTiC58 fragments were unable to replicate in Agrobacterium, these clones were found in Agrobacterium only if the acceptor carried a Ti plasmid, thus allowing a co-integration of the pBR322 clones with the Ti plasmid by homology recombination. These observations were used to develop an efficient method for site-specific mutagenesis of the Ti plasmids. pTiC58 fragnents, cloned in pBR322, were mutagenized in vitro and transformed into E. coli. The mutant clones were transmitted from an E. coli donor strain containing pGJ28 and R64drd11 to an Agrobacterium containing a target Ti plasmid. Selecting for stable transfer of the mutant clone utilizing its antibiotic resistance marker(s) gave exconjugants that already contained a co-integrate plasmid between the mutant clone and the Ti plasmid. A second recombination can dissociate the co-integrate plasmid into the desired mutant Ti plasmid and a non-replicating plasmid formed by the vector plasmid pBR322 and the target Ti fragment. These second recombinants lose the second plasmid and they are identified by screening for the appropriate marker combination.
Regulated gene expression of chimeric genes has been studied extensively in electroporated protoplasts. The applicability of these assays is limited, however, because protoplasts are not always physiologically identical to the cells from which they are derived. We have developed a procedure to electroporate DNA into intact and organized leaf structures of rice. Optimization of the new gene delivery system mainly involved eliminating explant-released nucleases, prolonging the DNA/explant incubation time, and expanding the pulse time. Using a [beta]-glucuronidase gene under the control of constitutive promoters, we demonstrated that all cell types within a leaf base were susceptible to electroporation-mediated DNA uptake. Although the technique was initially developed for leaf bases of young etiolated rice seedlings, we proved that it was equally applicable both to other monocotyledons, including wheat, maize, and barley, and to other explants, such as etiolated and green sheath and lamina tissues from rice. Transient gene expression assays with electroporated leaf bases showed that the promoter from a pea light-harvesting chlorophyll a/b-binding protein gene displayed both light- and chloroplast-dependent expression in rice, and that the promoter from the Arabidopsis S-adenosylmethionine synthetase gene was, as in transgenic Arabidopsis and tobacco, preferentially expressed in cells surrounding the vascular bundles.
Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.
We expressed the B subunit of enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1-ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the recombinant LTB protein, suggesting that it can be functional in prevention of diarrhea.
The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants.
To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM1-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.
We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S promoter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maximum of 0.3% LT-B in TAEP was measured in R3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional antigens.
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.
The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2,4-d) has been designed.The cells required thiamine, 2,4-d and ammonium in addition to the usual mineral salts and sucrose.Optimum concentrations of nitrate and ammonium were 25 and 2 mM respectively. The highest yield of cells was achieved at an initial pH of 4.5–5.5. During the growth cycle the pH gradually increased to 6.0–6.2.
The role of receptor binding in the toxicity, immunogenicity, and adjuvanticity of the heat-labile enterotoxin of Escherichia coli (LT) was examined by comparing native LT and LT(G33D), a B-subunit receptor binding mutant, with respect to the ability to bind to galactose and to GM1, toxicity on mouse Y-1 adrenal tumor cells, the ability to stimulate adenylate cyclase in Caco-2 cells, enterotoxicity in the patent mouse model, and oral immunogenicity and adjuvanticity. In contrast to native LT, LT(G33D) was unable to bind to the galactosyl moiety of Sepharose 4B or GM1 but did retain the lectin-like ability to bind to immobilized galactose on 6% agarose beads. LT(G33D) had no enterotoxicity in the patent mouse model but exhibited residual toxicity on mouse Y-1 adrenal tumor cells and had an ability equivalent to that of native LT to stimulate adenylate cyclase in Caco-2 cells (5,000 versus 6,900 pmol per mg of protein). In addition, LT(G33D) was unable to serve as an effective oral adjuvant for induction of immunoglobulin G or A directed against a coadministered antigen. Furthermore, LT(G33D) elicited negligible serum and mucosal antibody responses against itself. These data indicate that the toxicity, immunogenicity, and oral adjuvanticity of LT are dependent upon binding of the B subunit to ganglioside GM1.
A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)11 probe for the presence of (CA/GT)n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.
A gene encoding the mature Escherichia coli heat-labile enterotoxin B subunit (LTB) was introduced in a vector pNU212 and expressed at high levels in Bacillus brevis HPD31. The maximum amount of recombinant LTB (rLTB) secreted into the modified 5PY medium containing erythromycin was about 350 mg l(-1) when cultivated at 30 degrees C for 8 days. The rLTB purified directly from the culture supernatant by using D-galactose immobilized agarose was identical to the native LTB with respect to the molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the amino terminal amino acid sequence. Western blot analysis with antiserum to cholera toxin B subunit (CTB) indicated that rLTB had cross-reactivity to native CTB and its GM1 binding ability was almost the same as that of the CTB. The rLTB predominantly showed the pentameric form when non-boiled samples were applied to SDS-PAGE. When rLTB was administered intranasally to mice with diphtheria toxoid (D(T)), it resulted in the substantial stimulation of D(T)-specific serum IgG antibody, and in the induction of moderate levels of D(T)-specific mucosal IgA antibody responses in the nasal cavities and in the lung, suggesting that purified rLTB acts as a promising immunoadjuvant on mucosal immunizations.
Passive immunization plays an important role in protecting young mammals against pathogens before the maturation of their own immune systems. Although many reports have shown active immunization of animals and human through the use of plant-derived vaccines, only one report has given evidence of passive immunization of offspring through oral immunization of parents using plant-derived vaccines. In this case, a challenge alone provided the evidence of passive immunization and the mechanism through which this occurred was not investigated. This report describes the first step in elucidating the mechanism of passive immunization of offspring through actively immunizing the female parent through an orally delivered, plant-derived vaccine. The authors found passive immunization of offspring was caused by transfer of antigen-specific IgG through either transplacental transfer or ingesting colostrum. Future studies will investigate the roles of transplacental antibody transfer and ingesting colostrum in passive immunization and the possible involvement of IgA in this immunization route.
Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.
Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.
The B subunit of Escherichia coli heat-labile toxin (LTB) is a potent mucosal immunogen and immunoadjuvant for co-administered antigens. In order to produce large scale of LTB for the development of edible vaccine, we used transgenic somatic embryos of Siberian ginseng, which is known as medicinal plant. When transgenic somatic embryos were cultured in 130L air-lift type bioreactor, they were developed to mature somatic embryos through somatic embryogenesis and contained approximately 0.36% LTB of the total soluble protein. Enzyme-linked immunosorbent assay indicated that the somatic embryo-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting the LTB subunits formed active pentamers. Therefore, the use of the bioreactor system for expression of LTB proteins in somatic embryos allows for continuous mass production in a short-term period.
Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.
The B subunit of the heat labile toxin of enterotoxigenic Escherichia coli (LTB) was used as a model immunogen for production in soybean seed. LTB expression was directed to the endoplasmic reticulum (ER) of seed storage parenchyma cells for sequestration in de novo synthesized inert protein accretions derived from the ER. Pentameric LTB accumulated to 2.4% of the total seed protein at maturity and was stable in desiccated seed. LTB-soybean extracts administered orally to mice induced both systemic IgG and IgA, and mucosal IgA antibody responses, and was particularly efficacious when used in a parenteral prime-oral gavage boost immunization strategy. Sera from immunized mice blocked ligand binding in vitro and immunized mice exhibited partial protection against LT challenge. Moreover, soybean-expressed LTB stimulated the antibody response against a co-administered antigen by 500-fold. These results demonstrate the utility of soybean as an efficient production platform for vaccines that can be used for oral delivery.
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