Abstract
Vaccines produced by transgenic plants would have the potential to change the traditional means of production and inoculation of vaccines, and to reduce the cost of vaccine production. In the present study, an UreB antigen gene from a new Helicobacter pylori strain ZJC02 was cloned into the binary vector pBI121 which contains a CaMV35S promoter and a kanamycin resistance gene, and then transformed UreB into tobacco leaf-disc by Agrobacterium-mediated method. A total of 50 regenerated plants with kanamycin resistance were obtained in the selection media. The 35 putative transgenic individuals were tested and verified the presence and integration of the UreB into the nuclear genome of tobacco plants by PCR, PCR-southern, and Southern analyses. Expression of UreB gene in the tobacco plants was confirmed by RT-PCR and Western Blot analysis using polyclonal human antiserum. To our knowledge, this is the first report of the expression of Helicobacter pylori UreB antigen gene in a plant system, suggesting a major step in the production of plant-based vaccines for Helicobacter pylori.








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Arakawa T, Chong DKX, Langridge WHR (1998) Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nat Biotech 16:292–297
Arntzen CJ (1998) Pharmaceutical foodstuffs: oral immunization with transgenic plants. Nat Med 4:502–503
Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721
Blaser MJ, Berg DE (2001) Helicobacter pylori genetic diversity and risk of human disease. J Clin Invest 107:767–773
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Carrillo C, Wigdorovitz A, Oliveros JC, Zamorano PI, Sadir AM, Gómez N (1998) Protective immune response to foot and mouth disease virus with VP1 expressed in transgenic plants. J Virol 72:1688–1690
Castañón S, Marín MS, Martín Alonso JM, Boga JA, Casais R, Humara JM (1999) Immunization with potato plants expressing VP60 proteins protects against rabbit hemorragic disease virus. J Virol 73:4452–4455
Chiara P, Rossi M, Marchetti M (1997) Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection. Infect Immun 65:4996–5002
Clayton C, Wren BW, Mullany P, Topping A, Tabaqchali S (1989) Molecular cloning and expression of Campylobacter pylori species-specific antigens in Escherichia coli K-12. Infect Immun 57:623–629
Covacci A, Telford JL, Del Giudice G, Parsonnet J, Rappuoli R (1999) Helicobacter pylori virulence and genetic geography. Science 284:1328–1333
Del Giudice G (2001) The design of vaccines against Helicobacter pylori and their development. Annu Rev Immunol 19:523–563
De Wilde C, Podevin N, Windels P, Depicker A (2001) Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage effects. Mol Genet Genomics 265:647–653
Eaton KA, Brooks CL, Morgan DR, Krakowka S (1991) Essential role of urease on pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 59:2470–2475
Ferrero RL , Thiberge JM, Huerre M, Labigne A (1994) Recombinant antigens prepared from the urease subunits of Helicobacter pylori spp: evidence of protection in a mouse model of gastric infection. Infect Immun 62:4981–4989
Gómez-Duarte O, Lucas B, Yan ZX, Panthel K, Haas R, Meyer TF (1998) Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B. Vaccine 16:460–471
Gómez N, Carrillo C, Parra F, Salinas J, Borca MV, Escribano JM (1998) Expression of immunogenic glycoprotein-S-polypeptides from transmissible gastroenteritis coronavirus in transgenic plants. Virology 249:352–358
Gómez N, Wigdorovitz A, Castañón A, Gil F, Ordas R, Borca MV, et al. (2000) Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus. Arch Virol 145:1725–1732
Haq TA, Mason HS, Clements JD, Arntzen CJ (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268:714–716
Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfectionand transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187
Hood EE, Jilka JM (1999) Plant-based production of xenogenic proteins. Curr Opin Biotechnol 10(4):382–386
Horsch RB, Fry JE, Hofman NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
Hsun LM, Roussel Y, Wilks M, Tabaqchali S (2001) Expression of Helicobacter pylori urease subunit B gene in Lactococcus lactis MG(1363) and its use as a vaccine delivery system against H. pylori infection in mice. Vaccine 19:3927–3935
Kusnadi AR, Hood EE, Witcher DR, Howard JA, Nikolov ZL (1998) Production and purification of two recombinant proteins from transgenic corn. Biotechnol Prog 14:149–155
Marchetti M, Rossi M, Giannelli V, Giuliani MM, Pizza M, Censini S, Covacci A, Massari P, Pagliaccia C, Manetti R, Telford JL, Douce G, Dougan G, Rappuoli R, Ghiara P (1998) Protection against Helicobacter pylori infection in mice by intragastric vaccination with H. pylori antigens is achieved using a non-toxic mutant of E. coli heat-labile enterotoxin (LT) as adjuvant. Vaccine 16:33–37
Mason HS, Arntzen CJ (1995) Transgenic plants as vaccine production systems. Trends Biotechnol 13:388–392
Mason HS, Lam DMK, Arntzen CJ (1992) Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci USA 89:11745–11749
Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ (1996) Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc Natl Acad Sci USA 93:5335–5340
Mason HS, Haq TA, Clements JD, Arntzen CJ (1998) Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16:1335–1342
McKersie BD, Chen Y, de Beus M, Bowley SR (1993) Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiol 103:1155–1163
Mercenier A Wiedermann U, Breiteneder H (2001) Edible genetically modified microorganisms and plants for improved health. Curr Opin Biotechnol 12:510–515
Michetti P (1997) Vaccine against Helicobacter pylori: fact or fiction? Gut 41:728–730
Modelska A, Dietzschold B, Sleysh N (1998) Immunization against rabies with plant-derived antigen. Proc Natl Acad Sci USA 95:2481–2485
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Peach C, Velten J (1991) Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17:49–60
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Sheikh NA (2000) Delivery systems for molecular vaccination. Curr Opin Mol Ther 2:37–54
Telford JL, Ghiara P (1996) Prospect for the development of a vaccine against Helicobacter pylori. Drugs 52:799–804
Wang H, Qi M, Cutler AJ (1993) A simple method of preparing plant samples for PCR. Nucleic Acids Res 21:4153–4154
Warren JR, Marshall BJ (1983) Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1:1273–1275
Warzecha H, Mason HS (2003) Benefits and risks of antibody and vaccine production in transgenic. J Plant Physiol 160(7):755–764
Wigdorovitz A, Carrillo C, Dus Santos MJ, Sadir AM, R’ıos R, Franzione 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
Williams CL, Preston T, Hossack M, Slater C, McColl KE (1996) Helicobacter pylori utilizes urea for amino acid synthesis. FEMS Immunol Med Microbiol 13:87–94
Acknowledgements
The authors sincerely thank Dr. Chunyuan Huang (Adelaide University, Australia) for his critical reading of this manuscript. This work was supported by the National Natural Science Foundation of China (No. 30300017; 30370876), the Natural Science Foundation of Zhejiang Province (No. 301018), and the program for science and technology from Zhejiang Province (No. 2004C32015; 2003C34005).
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Gu, Q., Han, N., Liu, J. et al. Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.). Plant Cell Rep 24, 532–539 (2005). https://doi.org/10.1007/s00299-005-0962-8
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DOI: https://doi.org/10.1007/s00299-005-0962-8


