Improved expression of recombinant GFP using a replicating vector based on Beet curly top virus in leaf-disks and infiltrated Nicotiana benthamiana leaves

Kim, Kyung Il; Sunter, Garry; Bisaro, David M.; Chung, In Sik

doi:10.1007/s11103-007-9137-z

Cited by 31 publications

(20 citation statements)

References 51 publications

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“…, 2008). Likewise, a similar threefold increase in GFP had been observed in a previous study (Kim et al. , 2007) that used densitometry analysis of GFP on western blots to compare a replicating viral vector based on the geminivirus Beet curly top virus and a non‐replicating vector.…”

Section: Discussionsupporting

confidence: 75%

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

Regnard

Halley-Stott

Tanzer

et al. 2009

Plant Biotechnology Journal

126

108

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SummaryWe constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three-to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals.

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Section: Discussionsupporting

confidence: 75%

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

Regnard

Halley-Stott

Tanzer

et al. 2009

Plant Biotechnology Journal

126

108

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“…Geminiviral replicon systems have been used for plant production of several recombinant proteins, including GUS protein (Mor et al. , 2003), GFP (Kim et al. , 2007) and capsid proteins of hepatitis B and Norwalk viruses (Huang et al.…”

Section: Discussionmentioning

confidence: 99%

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

Phoolcharoen

Bhoo

Lai

et al. 2011

Plant Biotechnology Journal

141

131

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Summary Filoviruses (Ebola and Marburg viruses) cause severe and often fatal hemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identify Ebola and Marburg viruses as “category A” pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of Nicotiana benthamiana produced assembled immunoglobulin, which was purified by ammonium sulfate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine.

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“…The first recombinant HA-tagged PRRSV lost foreign HA epitope fused with the ORF7 from the second passage [43]. The rescued PRRSV with the green fluorescent gene fused with the Nsp2 protein, lost the green fluorescent after 7 serial passages due to the deletion of N-terminal amino acids (1 to 159aa) of GFP [44] or the accumulation of point mutations [45]. In addition, apart from the fusion of the green fluorescent gene with Nsp2, a recombinant PRRSV expressing GFP gene proved the stability of the foreign GFP gene even after 37 serial passages.…”

Section: Introductionmentioning

confidence: 99%

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit

Wang

Huang

Kong

et al. 2013

Vet Res

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Here we report the rescue of a recombinant porcine reproductive and respiratory syndrome virus (PRRSV) carrying an enhanced green fluorescent protein (EGFP) reporter gene as a separate transcription unit. A copy of the transcription regulatory sequence for ORF6 (TRS6) was inserted between the N protein and 3′-UTR to drive the transcription of the EGFP gene and yield a general purpose expression vector. Successful recovery of PRRSV was obtained using an RNA polymerase II promoter to drive transcription of the full-length virus genome, which was assembled in a bacterial artificial chromosome (BAC). The recombinant virus showed growth replication characteristics similar to those of the wild-type virus in the infected cells. In addition, the recombinant virus stably expressed EGFP for at least 10 passages. EGFP expression was detected at approximately 10 h post infection by live-cell imaging to follow the virus spread in real time and the infection of neighbouring cells occurred predominantly through cell-to-cell-contact. Finally, the recombinant virus generated was found to be an excellent tool for neutralising antibodies and antiviral compound screening. The newly established reverse genetics system for PRRSV could be a useful tool not only to monitor virus spread and screen for neutralising antibodies and antiviral compounds, but also for fundamental research on the biology of the virus.

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Improved expression of recombinant GFP using a replicating vector based on Beet curly top virus in leaf-disks and infiltrated Nicotiana benthamiana leaves

Cited by 31 publications

References 51 publications

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

A novel porcine reproductive and respiratory syndrome virus vector system that stably expresses enhanced green fluorescent protein as a separate transcription unit

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