Matching Items (30)

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Recombinant human osteopontin expressed in Nicotiana benthamiana stimulates osteogenesis related genes in human periodontal ligament cells

Description

Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell

Tissue engineering aims to utilise biologic mediators to facilitate tissue regeneration. Several recombinant proteins have potential to mediate induction of bone production, however, the high production cost of mammalian cell expression impedes patient access to such treatments. The aim of this study is to produce recombinant human osteopontin (hOPN) in plants for inducing dental bone regeneration. The expression host was Nicotiana benthamiana using a geminiviral vector for transient expression. OPN expression was confirmed by Western blot and ELISA, and OPN was purified using Ni affinity chromatography. Structural analysis indicated that plant-produced hOPN had a structure similar to commercial HEK cell-produced hOPN. Biological function of the plant-produced hOPN was also examined. Human periodontal ligament stem cells were seeded on an OPN-coated surface. The results indicated that cells could grow normally on plant-produced hOPN as compared to commercial HEK cell-produced hOPN determined by MTT assay. Interestingly, increased expression of osteogenic differentiation-related genes, including OSX, DMP1, and Wnt3a, was observed by realtime PCR. These results show the potential of plant-produced OPN to induce osteogenic differentiation of stem cells from periodontal ligament in vitro, and suggest a therapeutic strategy for bone regeneration in the future.

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Date Created
  • 2017-12-11

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Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves

Description

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

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Date Created
  • 2016-08-12

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5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

Description

We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus

We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.

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Date Created
  • 2016-02-24

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Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

Description

Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a

Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae) genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP) was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs). In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.

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Created

Date Created
  • 2014-05-11

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Production and functional testing of a recombinant fusion protein immunotherapy for glioblastoma

Description

Fusion protein immunotherapies such as the bispecific T cell engager (BiTE) have displayed promising potential as cancer treatments capable of engaging the immune system against tumor cells. It has been

Fusion protein immunotherapies such as the bispecific T cell engager (BiTE) have displayed promising potential as cancer treatments capable of engaging the immune system against tumor cells. It has been shown that chlorotoxin, a 36-amino peptide found in the venom of the deathstalker scorpion (Leiurus quinquestriatus), binds specifically to glioblastoma (GBM) cells without binding healthy tissue, making it an ideal GBM cell binding moiety for a BiTE-like molecule. However, chlorotoxin’s four disulfide bonds pose a folding challenge outside of its natural context and impede production of the recombinant protein in various expression systems, including those relying on bacteria and plants. To overcome this difficulty, we have engineered a truncated chlorotoxin variant (Cltx∆15) that contains just two of the original eight cystine residues, thereby capable of forming only a single disulfide bond while maintaining its ability to bind GBM cells. We further created a BiTE (ACDClx∆15) which tethers Cltx∆15 to a single chain ⍺-CD3 antibody in order to bring T cells into contact with GBM cells. The gene for ACDClx∆15 was cloned into a pET-11a vector for expression in Escherichia coli and isolated from inclusion bodies before purification via affinity chromatography. Immunoblot analyses confirmed that ACDClx∆15 can be expressed in E. coli and purified with high yield and purity; moreover, flow cytometry indicated that ACDClx∆15 is capable of binding GBM cells. These data warrant further investigation into the ability of ACDClx∆15 to activate T cells against GBM cells.

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Created

Date Created
  • 2019-05

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The Development of a Plant-Expressed M2e-Based Universal Influenza Vaccine

Description

Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins,

Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.

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Date Created
  • 2018-05

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Plant-derived Oral Vaccines: The Current State of the Science and Its Value for the Future

Description

The development of plant-derived antigens is very promising in vaccine research and the ability to synthesize vaccines cheaply and safely in plant, which can then be ingested, has enormous potential

The development of plant-derived antigens is very promising in vaccine research and the ability to synthesize vaccines cheaply and safely in plant, which can then be ingested, has enormous potential benefits. The goal of this project is to summarize and synthesize the work of current scientists on this issue into a cohesive argument in favor of plant-derived vaccinations, while acknowledging any possible drawbacks to their development and the actions that are being taken to overcome them. Hepatitis B, a virus for which orally administered, plant-based vaccines are currently being developed, serves as the case study in which these issues are analyzed. It was found that the synthesized protein is effective immunogenic in humans, but there is still the remaining challenge of making it generate a strong enough immune response through simple ingestion. For this reason, it is clear that plant-derived, oral vaccinations merit further research and hold real prospects of success.

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Date Created
  • 2008-05

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Expression and Purification of Viral Like Particles for Vaccines and Structural Analysis

Description

Succinylcholine-induced apnea is a common problem in pre-hospital medicine that affects 1/1800 patients who undergo rapid sequence intubation. Succinylcholine is an anesthetic that mimics the neurotransmitter, acetylcholine. It binds to

Succinylcholine-induced apnea is a common problem in pre-hospital medicine that affects 1/1800 patients who undergo rapid sequence intubation. Succinylcholine is an anesthetic that mimics the neurotransmitter, acetylcholine. It binds to cholinergic receptors, blocking acetylcholine access, and causes paralysis for (normally) only a short time. Butyrylcholinesterase, which is responsible for succinylcholine hydrolysis, is deficient in a small percentage of the population. Previous studies have shown that wild-type butyrylcholinesterase (BChE) can be produced in transient-expression Nicotiana benthamiana, and can reverse the effects of succinylcholine induced apnea through enzyme replacement therapy. The wild type enzyme is also capable of irreversibly binding and inactivating organophosphorus nerve agents and pesticides, and has also exhibited cocaine hydrolase activity. Super cocaine-hydrolyzing BChE mutants, which exceed 2000 times the catalytic capability of the wild-type, have been optimized and expressed in N. benthamiana. The purpose of this study was to determine whether these mutants also hydrolyze succinylcholine with improved efficiency. Variant 3 and Variant 4 exhibited catalytic efficiencies of 2.08 x 106 M-1 min-1 and 3.48 x 106 M-1 min-1, respectively, against their preferred substrate, butyrylthiocholine, in the Ellman assay. The wild-type plant-expressed BChE did exhibit hydrolysis of succinylcholine, as we had previously determined; however, neither Variant 3 nor Variant 4 demonstrated the ability to hydrolyze succinylcholine in our particular assay. Therefore, N. benthamiana-expressed Variant 3 and Variant 4 may not succeed as a dual treatment against cocaine toxicity and prolonged succinylcholine-induces paralysis.

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Date Created
  • 2017-05

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How the EnvZ/OmpR Two-component Regulatory System Affects fepA Gene Expression in Escherichia coli

Description

This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to

This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.

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Agent

Created

Date Created
  • 2016-05

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The Production of a Chimeric Monoclonal Antibody as a Therapeutic Agent Against Flaviviruses

Description

A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium

A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium tumefaciens infiltration. Characterization and expression studies were then conducted to confirm correct assembly of the antibody. Once the antibody was purified, an ELISA was conducted to validate that the antibody was able to bind to the flavivirus fusion loop.

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Agent

Created

Date Created
  • 2018-05