An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

Santi, L.; Batchelor, Lance A.; Huang, Zhong; Hjelm, Brooke E.; Kilbourne, Jacquelyn; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.

doi:10.1016/j.vaccine.2008.01.053

Cited by 190 publications

(184 citation statements)

References 35 publications

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“…Another advantage of using N. benthamiana as hosts for protein expression is the availability of a variety of expression vectors 2,5 . In this study, two deconstructed viral vectors, one based on a tobacco mosaic virus (TMV) RNA replicon system (MagnICON vectors) and the other derived from the bean yellow dwarf virus (BeYDV) DNA replicon system (geminiviral vectors) 4,11,[15][16][17][18] , are used to carry the GFP and DsRed gene and deliver them into N. benthamiana cells via A. tumefaciens. Three DNA constructs will be used for GFP or DsRed expression with MagnICON vectors.…”

Section: Introductionmentioning

confidence: 99%

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Leuzinger

Dent

Hurtado

et al. 2013

JoVE

Self Cite

158

157

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Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaledup for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education. Video LinkThe video component of this article can be found at

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Section: Introductionmentioning

confidence: 99%

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Leuzinger

Dent

Hurtado

et al. 2013

JoVE

Self Cite

158

157

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“…Since then, DNA of interest from different organisms has been delivered into plant cells through agroinfiltration, for a broad range of applications; far beyond studying plant-virus interactions [16]. The most popular method of agroinfiltration is "syringe agroinfiltration", involving the use of a needleless syringe to introduce Agrobacterium into plant leaves [18]. First, a small nick is created with a needle in the epidermis on the back side of the leaf, by gently scratching, without piercing through both sides ( Figure 1A).…”

Section: Agroinfiltration Methodsologies and Applicationsmentioning

confidence: 99%

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

Chen¹,

Lai²

2013

Adv Tech Biol Med

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111

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Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems, as a premier platform for commercial production of pharmaceutical proteins.

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“…KP881605.1) and CLCuBuV (GenBank accession no. HF567942) were constructed and checked their infectivity in tomato and tobacco plants along with CLCuMβ by using agroinfiltration method (Santi et al, 2008). Final sequences have already been submitted to the NCBI database.…”

Section: Selection/construction Of Infectious Clonesmentioning

confidence: 99%

Construction of an Infectious Chimeric Geminivirus by Molecular Cloning Based on Coinfection and Recombination

Khalid¹,

Zia-ur-Rehman²,

Ali³

et al. 2017

IJAB

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In the evolution of geminiviruses, genetic recombination plays a key role in ecological, biochemical and evolutionary processes. They evolve as results of prehistoric inter-genus recombination whereas intra-genus recombination of geminiviruses leads to the emergence of agriculturally important plant pathogens. Geminiviruses are transmitted by leafhoppers, treehoppers and whiteflies. An isolate of Chickpea chlorotic dwarf virus (CpCDV), a monopartite dicot infecting mastrevirus is transmitted by leafhopper. In contrast, Cotton leaf curl burewala virus (CLCuBuV), a monopartite begomovirus, was acquired and transmissible by whitefly to various host plants. In this study, we remove the coat protein (CP) gene of CpCDV by PCR and replaced with that of PCR amplified CLCuBuV-CP. The resultant infectious clone of chimeric CpCDV is produced later named mastrebegomo chimera (pGII0000MBC). By using Agroinfiltration technique (Agrobacterium-mediated inoculation) the chimeric clone along with wild type Clone CpCDV and CLCuBuV are injected on tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana benthamiana Domin.) seedlings and get symptoms after 21dpi (days post inoculation). Later, the symptoms appeared on plants were compared with healthy plants. This study elaborates our awareness about the genetic recombination and coexistence of mastreviruses with begomovirus globally including Pakistan and will provide the basic information regarding their management.

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An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

Cited by 190 publications

References 35 publications

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins

Construction of an Infectious Chimeric Geminivirus by Molecular Cloning Based on Coinfection and Recombination

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