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Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

Tinazzi E, Merlin M, Bason C, Beri R, Zampieri R, Lico C, Bartoloni E, Puccetti A, Lunardi C, Pezzotti M, Avesani L - Front Plant Sci (2015)

Bottom Line: Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms.We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients.Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Verona Verona, Italy.

ABSTRACT
Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

No MeSH data available.


Related in: MedlinePlus

Analysis of PVX nanoparticles. (A) Western blot of N. benthamiana leaf TSP extracts, 24 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-PVX CP antibody. C = non-infected leaf extract, 1 = PVX-lipo leaf extract, and 2 = PVX control leaf extract. (B) Silver staining of 750 ng purified particles separated by SDS-PAGE. 1 = PVX-lipo particles and 2 = PVX control particles. Arrows indicate the position of the 25 kDa molecular marker. (C) Transmission electron micrographs of purified PVX-lipo particles and (D) PVX control particles. Scale bar = 100 nm.
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Figure 1: Analysis of PVX nanoparticles. (A) Western blot of N. benthamiana leaf TSP extracts, 24 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-PVX CP antibody. C = non-infected leaf extract, 1 = PVX-lipo leaf extract, and 2 = PVX control leaf extract. (B) Silver staining of 750 ng purified particles separated by SDS-PAGE. 1 = PVX-lipo particles and 2 = PVX control particles. Arrows indicate the position of the 25 kDa molecular marker. (C) Transmission electron micrographs of purified PVX-lipo particles and (D) PVX control particles. Scale bar = 100 nm.

Mentions: The PVX-lipo VNPs were produced by introducing the lipocalin synthetic peptide sequence into the vector pPVXSma (Lico et al., 2006) to yield pPVXSma-lipo particles in which the peptide was fused to the N-terminus of a truncated CP and displayed on the particle surface. The peptide sequence was optimized for N. benthamiana codon usage, and virus movement was promoted by adding a serine residue between the initiator codon and the first native residue (Betti et al., 2012). The production of chimeric PVX particles depends on virus replication, so the genetic stability of the virus genome was verified after several re-infection cycles. The plants were infected using pPVXSma-lipo plasmid DNA to initiate the first cycle, followed thereafter by infection using sap from symptomatic leaves obtained 12 days after each round of infection. The chimeric CP gene remained stable through several passages as demonstrated by RT-PCR and sequencing (data not shown). The presence of PVX-lipo and wild-type PVX was verified by western blot analysis of infected leaf extracts (Figure 1A), where it is evident a single band corresponding to the viral CP, with a molecular weight shift in the PVX-lipo sample in comparison to wild-type particles, corresponding to the lipo peptide fused to the CP.


Plant-Derived Chimeric Virus Particles for the Diagnosis of Primary Sjögren Syndrome.

Tinazzi E, Merlin M, Bason C, Beri R, Zampieri R, Lico C, Bartoloni E, Puccetti A, Lunardi C, Pezzotti M, Avesani L - Front Plant Sci (2015)

Analysis of PVX nanoparticles. (A) Western blot of N. benthamiana leaf TSP extracts, 24 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-PVX CP antibody. C = non-infected leaf extract, 1 = PVX-lipo leaf extract, and 2 = PVX control leaf extract. (B) Silver staining of 750 ng purified particles separated by SDS-PAGE. 1 = PVX-lipo particles and 2 = PVX control particles. Arrows indicate the position of the 25 kDa molecular marker. (C) Transmission electron micrographs of purified PVX-lipo particles and (D) PVX control particles. Scale bar = 100 nm.
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Related In: Results  -  Collection

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Figure 1: Analysis of PVX nanoparticles. (A) Western blot of N. benthamiana leaf TSP extracts, 24 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-PVX CP antibody. C = non-infected leaf extract, 1 = PVX-lipo leaf extract, and 2 = PVX control leaf extract. (B) Silver staining of 750 ng purified particles separated by SDS-PAGE. 1 = PVX-lipo particles and 2 = PVX control particles. Arrows indicate the position of the 25 kDa molecular marker. (C) Transmission electron micrographs of purified PVX-lipo particles and (D) PVX control particles. Scale bar = 100 nm.
Mentions: The PVX-lipo VNPs were produced by introducing the lipocalin synthetic peptide sequence into the vector pPVXSma (Lico et al., 2006) to yield pPVXSma-lipo particles in which the peptide was fused to the N-terminus of a truncated CP and displayed on the particle surface. The peptide sequence was optimized for N. benthamiana codon usage, and virus movement was promoted by adding a serine residue between the initiator codon and the first native residue (Betti et al., 2012). The production of chimeric PVX particles depends on virus replication, so the genetic stability of the virus genome was verified after several re-infection cycles. The plants were infected using pPVXSma-lipo plasmid DNA to initiate the first cycle, followed thereafter by infection using sap from symptomatic leaves obtained 12 days after each round of infection. The chimeric CP gene remained stable through several passages as demonstrated by RT-PCR and sequencing (data not shown). The presence of PVX-lipo and wild-type PVX was verified by western blot analysis of infected leaf extracts (Figure 1A), where it is evident a single band corresponding to the viral CP, with a molecular weight shift in the PVX-lipo sample in comparison to wild-type particles, corresponding to the lipo peptide fused to the CP.

Bottom Line: Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms.We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients.Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Verona Verona, Italy.

ABSTRACT
Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX), chimeric virus particles, and Cowpea mosaic virus, empty virus-like particles to display a linear peptide (lipo) derived from human lipocalin, which is immunodominant in Sjögren's syndrome (SjS) and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

No MeSH data available.


Related in: MedlinePlus