Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants

Abstract

Chloroplast transformation has an extraordinary potential for antigen production in plants because of the capacity to accumulate high levels of recombinant proteins and increased biosafety due to maternal plastid inheritance in most crops. In this article, we evaluate tobacco chloroplasts transformation for the production of a highly immunogenic epitope containing amino acid residues 135–160 of the structural protein VP1 of the foot and mouth disease virus (FMDV). To increase the accumulation levels, the peptide was expressed as a fusion protein with the β-glucuronidase reporter gene (uidA). The recombinant protein represented the 51% of the total soluble proteins in mature leaves, a level higher than those of the Rubisco large subunit, the most abundant protein in the leaf of a wild-type plant. Despite this high accumulation of heterologous protein, the transplastomic plants and wild-type tobacco were phenotypically indistinguishable. The FMDV epitope expressed in transplastomic plants was immunogenic in mice. These results show that transplastomic tobacco express efficiently the recombinant protein, and we conclude that this technology allows the production of large quantities of immunogenic proteins.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

FMDV:

Foot and mouth disease virus

ELISA:

Enzyme-linked immunosorbent assay

TSP:

Total soluble proteins

References

  1. Bachrach H (1977) Foot and mouth disease virus: properties, molecular biology and immunogenicity. In: Romberger (ed) Betsville symposia in agricultural research I. Virology in agriculture. Allanheld-Osmiun, Montclair, NY, pp 3–32

  2. Bock R (2007) Plastid biotechnology: prospect for herbicide and insect resistance, metabolic engineering and molecular farming. Curr Opin Biotechnol 18:100–106

  3. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  4. Chebolu S, Daniell H (2007) Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in transgenic chloroplasts and immunogenicity in mice towards vaccine development for amoebiasis. Plant Biotechnol J 5:230–239

  5. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995

  6. Daniell H (2006) Production of biopharmaceuticals and vaccines in plants via the chloroplast genome. Biotechnol J 1:1071–1079

  7. Daniell H, Lee SB, Panchal T, Wiebe PO (2001) Expression of cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol Biol 311:1001–1009

  8. De Cosa B, Moar W, Lee S, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74

  9. Dellaporta SL, Chomet PS, Mottinger JP, Wood JA, Yu SM, Hicks JB (1984) Endogenous transposable elements associated with virus infection in maize. Cold Spring Harb Symp Quant Biol 49:321–328

  10. Dus Santos MJ, Wigdorovitz A, Trono K, Ríos RD, Franzone PM, Gil F, Moreno J, Carrillo C, Escribano JM, Borca MV (2002) A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine 20:1141–1147

  11. Fernández-San Millan A, Ortigosa SM, Hervas-Stubbs S, Corral-Martinez P, Segui-Simarro JM, Gaetan J, Coursaget P, Veramendi J (2008) Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol J 6:427–441

  12. Floss DM, Falkenburg D, Conrad U (2007) Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview. Trans Res 16:315–332

  13. Grubman MJ, Baxt B (2004) Foot and mouth. Dis Clin Microbiol Rev 17:465–493

  14. Hager M, Biehler K, Illerhaus J, Ruf S, Bock R (1999) Targeted inactivation of the smallest plastid genome-encoded open reading frame reveals a novel and essential subunit of the cytochrome b(6)f complex. EMBO J 18(21):5834–5842

  15. Herz S, Füßl M, Steiger S, Koop H (2005) Development of novel types of plastid transformation vectors and evaluation of factors controlling expression. Trans Res 14:969–982

  16. Hondred D, Walker JM, Mathews DE, Vierstra RD (1999) Use of ubiquitin fusions to augment protein expression in transgenic plants. Plant Physiol 119:713–723

  17. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS-fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

  18. Joensuu JJ, Kiklander-Teeri V, Brandle JE (2008) Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control. Phytochem Rev 7:553–577

  19. Koya V, Moayeri M, Leppla SH, Daniell H (2005) Plant based vaccine: mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infect Immun 73:8266–8274

  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  21. LaVallie ER, McCoy JM (1995) Gene fusion expression systems in Escherichia coli. Curr Opin Biotechnol 6:501–506

  22. Leelavathi S, Reddy VS (2003) Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol Breed 11:49–58

  23. Li Y, Sun M, Liu J, Yang Z, Zhang Z, Shen G (2005) High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts. Plant Cell Rep 25(4):329–333

  24. Mc Cabe MS, Klaas M, Gonzalez-Rabade N, Poage M, Badillo-Corona JA, Zhou F, Karcher D, Bock R, Gray JC, Dix PJ (2008) Plastid transformation of high-biomass tobacco variety Maryland Mammoth for production of human immunodeficiency virus type 1 (HIV-1) p24 antigen. Plant Biotechnol J 6:914–929

  25. Molina A, Hervás-Stubbs S, Daniell H, Mingo-Castel AM, Veramendi J (2004) High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnol J 2:141–153

  26. Oey M, Lohse M, Kreikemeyer B, Bock R (2009) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445

  27. Ruf S, Biehler K, Bock R (2000) A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna. J Cell Biol 149(2):369–378

  28. Rybicki EP (2009) Plant-produced vaccines: promise and reality. Drug Disc Today 14:16–24

  29. Sobrino F, Sáiz M, Jiménez-Clavero MA, Núñez JI, Rosas MF, Baranowski E, Ley V (2001) Foot-and-mouth disease virus: a long known virus, but a current threat. Vet Res 32:1–30

  30. Svab Z, Maliga P (1993) High-frequency plastid transformation by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917

  31. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastid in higher plants. Proc Natl Acad Sci USA 87:8526–8530

  32. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin fragment C in tobacco chloroplasts. Nucleic Acids Res 31:1174–1179

  33. Vermij P, Waltz E (2006) USDA approves the first plant-based vaccine. Nat Biotechnol 24:233–234

  34. Wigdorovitz A, Carrillo C, Dus Santos MJ, Sadir AM, Ríos R, Franzone P (1999) Induction of a protective antibody response to foot and mouth disease virus in mice following oral or parenteral immunized with alfalfa transgenic plants expressing the viral structural protein VP1. Virology 255:347–353

  35. Wirth S, Segretin ME, Mentaberry A, Bravo-Almonacid FF (2006) Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light. J Biotechnol 125:159–172

  36. Zhou F, Badillo-Corona JA, Karcher D, Gonzalez-Rabade N, Piepenburg K, Borchers AM, Maloney AP, Kavanagh TA, Gray JC, Bock R (2008) High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes. Plant Biotechnol J 6:897–913

Download references

Acknowledgments

We wish to thank Demian Bellido and Fatima Torales for their technical assistance and Carolina Pometti for carefully reading the manuscript. This work has been supported by Instituto Nacional de Tecnología Agropecuaria (INTA-Argentina), project “Fiebre Aftosa”, no. AESA 1572. EML and MES are fellows of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-Argentina). MMM is assistant teacher of Universidad de Buenos Aires, Argentina. FBA and SAW are research scientists of CONICET, MJDS and AW are research scientists of CONICET and INTA, and MVM is research scientist of INTA.

Author information

Correspondence to Fernando Félix Bravo-Almonacid.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lentz, E.M., Segretin, M.E., Morgenfeld, M.M. et al. High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants. Planta 231, 387 (2010). https://doi.org/10.1007/s00425-009-1058-4

Download citation

Keywords

  • Antigen
  • Foot and mouth disease virus
  • Molecular farming
  • Plastid transformation
  • Tobacco