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Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization

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Abstract

Recently we obtained complexes between genetically modified Tobacco Mosaic Virus (TMV) particles and proteins carrying conserved influenza antigen such as M2e epitope. Viral vector TMV-N-lys based on TMV-U1 genome was constructed by insertion of chemically active lysine into the exposed N-terminal part of the coat protein. Nicotiana benthamiana plants were agroinjected and TMV-N-lys virions were purified from non-inoculated leaves. Preparation was analyzed by SDS-PAGE/Coomassie staining; main protein with electrophoretic mobility of 21 kDa was detected. Electron microscopy confirmed the stability of modified particles. Chemical conjugation of TMV-N-lys virions and target influenza antigen M2e expressed in E. coli was performed using 5 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 1 mM N-hydroxysuccinimide. The efficiency of chemical conjugation was confirmed by Western blotting. For additional characterization we used conventional electron microscopy. The diameter of the complexes did not differ significantly from the initial TMV-N-lys virions, but complexes formed highly organized and extensive network with dense “grains” on the surface. Dynamic light scattering demonstrated that the single peaks, reflecting the complexes TMV-N-lys/DHFR-M2e were significantly shifted relative to the control TMV-N-lys virions. The indirect enzymelinked immunosorbent assay with TMV- and DHFR-M2e-specific antibodies showed that the complexes retain stability during overnight adsorption. Thus, the results allow using these complexes for immunization of animals with the subsequent preparation of a candidate universal vaccine against the influenza virus.

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Abbreviations

a.a.:

amino acid residue

ADFK:

alanine-aspartic acid-phenylalanine-lysine sequence

CP:

coat protein

DHFR:

dihydrofolate reductase

EDC:

1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide

M2e:

conserved epitope of influenza A virus (25.5 kDa)

NHS:

N-hydroxysuccinimide

NP:

nucleoprotein

TMV:

tobacco mosaic virus

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Authors and Affiliations

  1. Faculty of Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    T. V. Gasanova, A. A. Koroleva, E. V. Skurat & P. A. Ivanov

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  1. T. V. Gasanova
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  2. A. A. Koroleva
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  3. E. V. Skurat
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  4. P. A. Ivanov
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Correspondence to T. V. Gasanova.

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Ethical approval. This article does not contain description of studies with human participants or animals performed by any of the authors.

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Conflict of interest. The authors declare no conflict of interest in financial or any other sphere.

Russian Text © The Author(s), 2020, published in Biokhimiya, 2020, Vol. 85, No. 2, pp. 260-271.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM19-137, December 30, 2019.

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Gasanova, T.V., Koroleva, A.A., Skurat, E.V. et al. Complexes Formed via Bioconjugation of Genetically Modified TMV Particles with Conserved Influenza Antigen: Synthesis and Characterization. Biochemistry Moscow 85, 224–233 (2020). https://doi.org/10.1134/S0006297920020091

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