Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana

Regnard, Guy L.; Rybicki, Edward P.; Hitzeroth, Inga I.

doi:10.1186/s12985-017-0847-9

Cited by 10 publications

(6 citation statements)

References 62 publications

(80 reference statements)

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“…This is comparable to reported purified yields in E. coli of 3 mg per litre of culture (Trundova and Celer, ). The PCV‐2 CP was targeted to the cytosol, but total protein yield could potentially be further optimized by targeting the proteins to various cellular organelles: this has previously been demonstrated for the accumulation of plant‐produced CP for the parrot‐infecting beak and feather disease virus (BFDV; Regnard et al ., ) and for human papillomavirus L1 CP (Maclean et al ., ). The CsCl gradient profile obtained here showed the plant‐produced PCV‐2 CP had a buoyant density of 1.564 g/cm 3 : this differs to previous reports for PCV, which report 1.37 g/cm 3 (Allan and Ellis, ; Liu et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Gunter

Regnard

Rybicki

et al. 2019

Plant Biotechnology Journal

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Summary Porcine circovirus type 2 ( PCV ‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease ( PCAD ). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV ‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV ‐2 capsid protein ( CP ) from plants is an essential first step towards the goal of a plant‐produced PCV ‐2 vaccine candidate. In this study, the PCV ‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV ‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles ( VLP s) resembling native virions and up to 6.5 mg of VLP s could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV ‐2 VLP s elicited specific antibody responses to PCV ‐2 CP . This is the first report describing the expression of PCV ‐2 CP in plants, the confirmation of its assembly into VLP s and the demonstration of their use to elicit a strong immune response in a mammalian model.

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Section: Discussionmentioning

confidence: 99%

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Gunter

Regnard

Rybicki

et al. 2019

Plant Biotechnology Journal

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“…Syringe agroinfiltration is a rapid method of introducing the target gene into the plant cells with no need for specialized instruments [26,28]. This method often results in the same [49] or even higher [50] levels of expression than the vacuum infiltration method, which allows for a more precise assessment of the expressed protein. Furthermore, the efficiency of two infiltration buffers (PBS-A and MMS-A) on transient expression of IHNV-G protein in bean leaves was studied.…”

Section: Discussionmentioning

confidence: 99%

Transient expression of the full‐length glycoprotein from infectious hematopoietic necrosis virus in bean (Phaseolus vulgaris) leaves via agroinfiltration

Fazeli

Golkar

Mirakhorli

et al. 2020

Biotech and App Biochem

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The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO 4 + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 μg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.

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“…However, it is only recently that VLPs have been made for either virus in plants. BFDV CP was successfully expressed in N. benthamiana via an Agrobacterium ‐delivered geminivirus‐derived replicating vector: the protein assembled into regular VLPs that could be purified by cesium chloride gradient centrifugation and appeared identical to native virions (Regnard, Rybicki, & Hitzeroth, ). A similar expression strategy for PCV‐2 CP resulted in yields of purified VLPs of up to 6.5 mg/kg, which could be purified by simple centrifugation techniques and were highly immunogenic when injected into mice (Gunter, Regnard, Rybicki, & Hitzeroth, ).…”

Section: Animal Virus‐derived Vnpsmentioning

confidence: 99%

Plant molecular farming of virus‐like nanoparticles as vaccines and reagents

Rybicki

2019

WIREs Nanomed Nanobiotechnol

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The use of plants for the production of virus‐like nanoparticles (VNPs) dates back to separating natural empty capsids of plant viruses from whole virions nearly 70 years ago, through to the present use of transgenic plants or recombinant Agrobacterium tumefaciens and/or plant virus‐derived vectors for the transient expression of engineered viral or other structural proteins in plants—a production system also known as molecular farming. Plant production of heterologous proteins has major advantages in terms of convenience—whole plants are generally used, and processes do not need to be sterile—and cost, as bulk biomass production is significantly cheaper than by any other method. Plant‐made VNPs in current use for nanotechnology include whole virions and naturally occurring empty capsids of plant viruses, and particles made by reassembly of coat protein (CP) purified from virions or by recombinant expression. Engineered VNP‐forming animal or human virus CPs expressed in plants include L1 protein from human papillomaviruses, human norovirus CP, hepatitis B surface and core antigens, influenza virus HA protein and HIV Gag polyprotein forming large enveloped particles by budding, orbi‐ and rotavirus particles that require assembly of four co‐expressed proteins, and polio‐ and foot and mouth disease viruses which require proteolytic processing of a polyprotein precursor to form 4‐component VNPs. Both plant and animal virus‐derived plant‐made VNPs can be used for surface and internal display of heterologous peptides or even whole proteins. A significant recent development has been the production of pseudovirions in plants, comprising plant or animal virus CPs and RNA or DNA pseudogenomes that can be used to deliver nucleic acid payloads into cultured cells or specific tissues or tumors in whole animals. This article is characterized under: Biology‐Inspired Nanomaterials > Protein and Virus‐Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies Diagnostic Tools > in vivo Nanodiagnostics and Imaging

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Recombinant expression of beak and feather disease virus capsid protein and assembly of virus-like particles in Nicotiana benthamiana

Cited by 10 publications

References 62 publications

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Transient expression of the full‐length glycoprotein from infectious hematopoietic necrosis virus in bean (Phaseolus vulgaris) leaves via agroinfiltration

Plant molecular farming of virus‐like nanoparticles as vaccines and reagents

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