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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 and characterization of CPMV eVLPs. (A) Western blot of N. benthamiana leaf TSP extracts, 11 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-CPMV CP antibody. C = non-infected leaf extract, 1 = CPMV-lipo leaf extract, and 2 = CPMV control leaf extract. (B) Silver staining of 2 μg purified particles separated by SDS-PAGE. 1 = CPMV-lipo particles and 2 = CPMV control particles. Arrows indicate the position of the 25 and 35 kDa molecular markers. (C) DLS analysis comparing purified CPMV-lipo (blue line) to unmodified CPMV (red line).
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Figure 2: Analysis and characterization of CPMV eVLPs. (A) Western blot of N. benthamiana leaf TSP extracts, 11 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-CPMV CP antibody. C = non-infected leaf extract, 1 = CPMV-lipo leaf extract, and 2 = CPMV control leaf extract. (B) Silver staining of 2 μg purified particles separated by SDS-PAGE. 1 = CPMV-lipo particles and 2 = CPMV control particles. Arrows indicate the position of the 25 and 35 kDa molecular markers. (C) DLS analysis comparing purified CPMV-lipo (blue line) to unmodified CPMV (red line).

Mentions: The CPMV-lipo eVLPs were produced using the pEAQ-HT system (Sainsbury et al., 2009). The VP60 CP precursor and the 24K protease were expressed on two separate pEAQ constructs allowing the production of eVLPs structurally identical to wild-type CPMV particles (Saunders et al., 2009). The sequence encoding the target peptide was inserted into vector pEAQ-HT-VP60, within the βB–βC loop of the small CP. N. benthamiana leaves were agroinfiltrated simultaneously with both vectors. Leaf extracts were prepared 6 dpi for analysis by western blot using anti-CPMV antibodies, revealing the presence of specific signals corresponding to the large and small and a molecular weight shift accounting for the modified small CP, thus confirming the insertion of the foreign peptide (Figure 2A).


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 and characterization of CPMV eVLPs. (A) Western blot of N. benthamiana leaf TSP extracts, 11 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-CPMV CP antibody. C = non-infected leaf extract, 1 = CPMV-lipo leaf extract, and 2 = CPMV control leaf extract. (B) Silver staining of 2 μg purified particles separated by SDS-PAGE. 1 = CPMV-lipo particles and 2 = CPMV control particles. Arrows indicate the position of the 25 and 35 kDa molecular markers. (C) DLS analysis comparing purified CPMV-lipo (blue line) to unmodified CPMV (red line).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Analysis and characterization of CPMV eVLPs. (A) Western blot of N. benthamiana leaf TSP extracts, 11 μl per lane separated by SDS-PAGE and detected with an alkaline phosphatase conjugated anti-CPMV CP antibody. C = non-infected leaf extract, 1 = CPMV-lipo leaf extract, and 2 = CPMV control leaf extract. (B) Silver staining of 2 μg purified particles separated by SDS-PAGE. 1 = CPMV-lipo particles and 2 = CPMV control particles. Arrows indicate the position of the 25 and 35 kDa molecular markers. (C) DLS analysis comparing purified CPMV-lipo (blue line) to unmodified CPMV (red line).
Mentions: The CPMV-lipo eVLPs were produced using the pEAQ-HT system (Sainsbury et al., 2009). The VP60 CP precursor and the 24K protease were expressed on two separate pEAQ constructs allowing the production of eVLPs structurally identical to wild-type CPMV particles (Saunders et al., 2009). The sequence encoding the target peptide was inserted into vector pEAQ-HT-VP60, within the βB–βC loop of the small CP. N. benthamiana leaves were agroinfiltrated simultaneously with both vectors. Leaf extracts were prepared 6 dpi for analysis by western blot using anti-CPMV antibodies, revealing the presence of specific signals corresponding to the large and small and a molecular weight shift accounting for the modified small CP, thus confirming the insertion of the foreign peptide (Figure 2A).

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