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ISSN 1027-2852 (Electronic)
ISSN 0864-4551 (Print)

2016, Number 2

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Biotecnol Apl 2016; 33 (2)

Expression of the recombinant tE2 antigen of bovine viral diarrhea virus (BVDV) in Nicotiana tabacum plants and biosafety studies in mice

Laguía M, Gallo M, Rubio C, Langle Y, Martinez S, Sanjuan P, Sanjuan N, Alvarez MA, Benavides P, Marconi PL

Language: English
References: 28
Page: 2221-2226
PDF size: 640.97 Kb.


ABSTRACT

Antigenic glycoprotein tE2 from bovine viral diarrhea virus (BVDV) and the fluorescence marker EGFP were transiently expressed in leaves of Nicotiana tabacum using Agrobacterium tumefaciens as vector. Analyzing the Agrobacterium and EGFP RNA/protein persistence allowed us to estimate an environmental exposure to recombinant material during 6 days after agroinfiltration. Time course analysis for the expression of EGFP shows silencing after 6 days post-infiltration with recombinant Agrobacterium strain in treated leaves. The leaf extracts expressing tE2 and EGFP were clarified at 6 days after agroinfiltration. The extracts were mixed with oil or aqueous adjuvants and injected to Balb/c mice in order to establish if the plant extracts are innocuous. Four doses were applied, followed by necropsy. No significant differences between non-recombinant extracts (control treatment) or recombinant leaf extracts in clinical and biochemical analysis were observed. In all mice injected, the adjuvant caused the classical pathological lesions observed in typical inflammatory processes at the site of inoculation. Histological sections of these lesions and major organs showed no further injury.


REFERENCES

  1. Sack M, Hofbauer A, Fischer R, Stoger E. The increasing value of plantmade proteins. Curr Opin Biotechnol. 2015;32:163-70.

  2. Tremblay R, Wang D, Jevnikar AM, Ma S. Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv. 2010;28(2):214-21.

  3. Chen Q, Lai H, Hurtado J, Stahnke J, Leuzinger K, Dent M. Agroinfiltration as an Effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Adv Tech Biol Med. 2013; 1:103.

  4. Nelson G, Marconi P, Periolo O, La Torre J, Alvarez MA. Immunocompetent truncated E2 glycoprotein of bovine viral diarrhea virus (BVDV) expressed in Nicotiana tabacum plants: a candidate antigen for new generation of veterinary vaccines. Vaccine. 2012;30(30):4499-504.

  5. Nielsen KM, Johnsen PJ, Bensasson D, Daffonchio D. Release and persistence of extracellular DNA min the environment. Env Biosafety Res. 2007;6(1-2):37-53.

  6. Brenner DJ, Krieg NR, Staley JR. Bergey’s Manual® of Systematic Bacteriology: Volume 2: The Proteobacteria, Part B: The Gammaproteobacteria. New York: Springer Science & Business Media; 2007, p. 1106.

  7. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82.

  8. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54.

  9. Marzocca MP, Seki C, Giambiagi SM, Robiolo B, Schauer R, Dus Santos MJ, et al. Truncated E2 of bovine viral diarrhea virus (BVDV) expressed in Drosophila melanogaster cells: a candidate antigen for a BVDV ELISA. J Virol Methods. 2007;144(1- 2):49-56.

  10. Suckow MA, Danneman P, Brayton C. The laboratory mouse. Boca Raton: CRC Press; 2001.

  11. Sparrow P, Broer I, Hood EE, Eversole K, Hartung F, Schiemann J. Risk assessment and regulation of molecular farming - a comparison between Europe and US. Curr Pharm Des. 2013;19(31):5513-30.

  12. Imlau A, Truernit E, Sauer N. Cellto-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell. 1999;11(3):309-22.

  13. Mishra NS, Mukherjee SK. A Peep into the plant miRNA World. Open Plant Sci J. 2007;1:1-9.

  14. Chau BL, Lee KA. Function and anatomy of plant siRNA pools derived from hairpin transgenes. Plant Methods. 2007;3:13.

  15. Walter A, Liebisch F, Hund A. Plant phenotyping: from bean weighing to image analysis. Plant Methods. 2015;11:14.

  16. Wirth-Dzięciołowska E, Karaszewska J, Sadowski T, Pyśniak K, Gajewska M. Selected blood serum biochemical indicators in twelve inbred strains of laboratory mice. Animal Sc Papers Reps. 2009;27(2):159-67.

  17. Goto N, Kato H, Maeyama J, Shibano M, Saito T, Yamaguchi J, et al. Local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties. Vaccine. 1997;15(12-13):1364-71.

  18. Aucouturier J, Dupuis L, Ganne V. Adjuvants designed for veterinary and human vaccines. Vaccine. 2001;19(17-19):2666-72.

  19. Fox CB, Haensler J. An update on safety and immunogenicity of vaccines containing emulsion-based adjuvants. Expert Rev Vaccines. 2013;12(7):747-58.

  20. Littleton LC, Gruenke JA. A comparison of the effect of three adjuvants on the antibody response to ovalbumin in mice. BIOS. 2013;84(3):142-7.

  21. Dey AK, Malyala P, Singh M. Physicochemical and functional characterization of vaccine antigens and adjuvants. Expert Rev Vaccines. 2014;13(5):671-85.

  22. De Gregorio E, Tritto E, Rappuoli R. Alum adjuvanticity: unraveling a century old mystery. Eur J Immunol. 2008;38(8):2068-71.

  23. Kleinau S, Erlandsson H, Holmdahl R, Klareskog L. Adjuvant oils induce arthritis in the DA rat. I. Characterization of the disease and evidence for an immunological involvement. J Autoimmun. 1991;4(6):871-80.

  24. Scragg AH. Bioreactor for the mass cultivation of cell plants. In: Plant Biotechnology. Eds. Fowler, MW and Waren, GS. London: Pergamon Press; 1992. p. 45-60.

  25. Marconi PL, Klykov S. Chapter 9: Mathematical modeling in recombinant plant systems: The challenge to produce heterologous proteins under GLP/GMP. In: MA Alvarez, Ed. Plant Biotechnology for Health: from secondary metabolites to molecular farming. New York: Springer; 2014. p. 145-55.

  26. Berinstein A, Vazquez-Rovere C, Asurmendi S, Gomez E, Zanetti F, Zabal O, et al. Mucosal and systemic immunization elicited by Newcastle disease virus (NDV) transgenic plants as antigens. Vaccine. 2005;23(48- 49):5583-9.

  27. Clemente M, Curilovic R, Sassone A, Zelada A, Angel SO, Mentaberry AN. Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity. Mol Biotechnol. 2005;30(1):41-50.

  28. Yácono MDL, Farran I, Becher ML, Sander V, Sanchez VR, Martin V, et al. A chloroplastderived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice. Plant Biotechnol J. 2012;10(9):1136-44.




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