Advertisement

Molecular Biology Reports

, Volume 45, Issue 6, pp 2237–2246 | Cite as

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato

  • Jafari Davod
  • Dehghan Nayeri FatemehEmail author
  • Hossein Honari
  • Ramin Hosseini
Original Article

Abstract

Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis. PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals.

Keywords

Anthrax Cholera Tomato Edible vaccine Shigellosis Agro-infiltration 

Notes

Acknowledgement

This work was done in Agricultural Biotechnology Department of Imam Khomeini International University. We appreciate all staffs on their good collaborations.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

Ethical approval and informed consent were not required for this type of study.

Research involving human participants or animals

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

References

  1. 1.
    Gangarosa EJ, Perera DR, Mata LJ, Morris CM, Guzman G, Reller LB (1970) Epidemic Shiga bacillus dysentery in Central America. II. Epidemiologic studies in 1969. J Infect Dis 122:181–190CrossRefGoogle Scholar
  2. 2.
    Sansonetti PJ (2001) Rupture, invasion and inflammatory destruction of the intestinal barrier by Shigella, making sense of prokaryote–eukaryote cross-talks. FEMS Microbiol Rev 25:3–14PubMedGoogle Scholar
  3. 3.
    Sasakawa C, Kamata K, Sakai T, Murayama SY, Makino S, Yoshikawa M (1986) Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun 51:470–475PubMedPubMedCentralGoogle Scholar
  4. 4.
    Lisanby MW (2009) Examination of the capacity of cathelicidins to control Bacillus anthracis pathogenesis. ProQuest, Ann ArborGoogle Scholar
  5. 5.
    Burnett J (1991) Anthrax. Cuitis 48:113–114Google Scholar
  6. 6.
    Longfield R (1991) Anthrax. In: Strickland GT (ed) Hunter’s tropical medicine. WB Saunders, Philadelphia, pp 434–438Google Scholar
  7. 7.
    Lakshmi N, Kumar A (1992) An epidemic of human anthrax—a study. Indian J Pathol Microbiol 35:1–4PubMedGoogle Scholar
  8. 8.
    Leppla SH (1982) Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci USA 79:3162–3166CrossRefGoogle Scholar
  9. 9.
    Hoover D, Friedlander A, Rogers L, Yoon I, Warren R, Cross A (1994) Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP. Infect Immun 62:4432–4439PubMedPubMedCentralGoogle Scholar
  10. 10.
    Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, Ahn NG, Oskarsson MK, Fukasawa K, Paull KD (1998) Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280:734–737CrossRefGoogle Scholar
  11. 11.
    Vitale G, Pellizzari R, Recchi C, Napolitani G, Mock M, Montecucco C (1998) Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun 248:706–711CrossRefGoogle Scholar
  12. 12.
    Molloy S, Bresnahan P, Leppla SH, Klimpel K, Thomas G (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-XX-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402PubMedGoogle Scholar
  13. 13.
    Elliott JL, Mogridge J, Collier RJ (2000) A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen. Biochemistry 39:6706–6713CrossRefGoogle Scholar
  14. 14.
    Mogridge J, Cunningham K, Lacy DB, Mourez M, Collier RJ (2002) The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen. Proc Natl Acad Sci USA 99:7045–7048CrossRefGoogle Scholar
  15. 15.
    Beauregard KE, Collier RJ, Swanson JA (2000) Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization. Cell Microbiol 2:251–258CrossRefGoogle Scholar
  16. 16.
    Milne JC, Collier RJ (1993) pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen. Mol Microbiol 10:647–653CrossRefGoogle Scholar
  17. 17.
    Blaustein RO, Koehler TM, Collier RJ, Finkelstein A (1989) Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers. Proc Natl Acad Sci USA 86:2209–2213CrossRefGoogle Scholar
  18. 18.
    Wesche J, Elliott JL, Falnes P, Olsnes S, Collier RJ (1998) Characterization of membrane translocation by anthrax protective antigen. Biochemistry 37:15737–15746CrossRefGoogle Scholar
  19. 19.
    Arêas APM, Oliveira MLS, Miyaji EN, Leite LCC, Aires KA, Dias WO, Ho PL (2004) Expression and characterization of cholera toxin B—pneumococcal surface adhesin A fusion protein in Escherichia coli: ability of CTB-PsaA to induce humoral immune response in mice. Biochem Biophys Res Commun 321:192–196CrossRefGoogle Scholar
  20. 20.
    Odumosu O, Nicholas D, Yano H, Langridge W (2010) AB toxins: a paradigm switch from deadly to desirable. Toxins 2:1612–1645CrossRefGoogle Scholar
  21. 21.
    Xu X, Gan Q, Clough RC, Pappu KM, Howard JA, Baez JA, Wang K (2011) Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylas. BMC Biotechnol 11:69CrossRefGoogle Scholar
  22. 22.
    De Muynck B, Navarre C, Boutry M (2010) Production of antibodies in plants: status after twenty years. Plant Biotechnol J 8:529–563CrossRefGoogle Scholar
  23. 23.
    Giddings G (2001) Transgenic plants as protein factories. Curr Opin Biotechnol 12:450–454CrossRefGoogle Scholar
  24. 24.
    Sambrook J, Russell DWS (2006) The condensed protocols from molecular cloning: a laboratory manualGoogle Scholar
  25. 25.
    Hesaraki M, Saadati M, Honari H, Olad G, Heiat M, Malaei F, Ranjbar R (2013) Molecular cloning and biologically active production of IpaD N-terminal region. Biologicals 41:269–274CrossRefGoogle Scholar
  26. 26.
    Finlay BB, Falkow S (1997) Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61:136–169PubMedPubMedCentralGoogle Scholar
  27. 27.
    Safaei S, Honari H, Mousavy SJ, Esmaeili A, Ghofrani M (2013) Isolation, cloning and fusion of N-terminal region of ipaD Shigella dysenteriea and Ricin toxin B subunit. Genet 3rd Millenn 10:2880–2889Google Scholar
  28. 28.
    Honari H, Amlashi I, Minaee ME, Safaee S (2013) Immunogenicity in Guinea pigs by IpaD-STxB recombinant protein. HBI J 16:83–93Google Scholar
  29. 29.
    Heine SJ, Diaz-McNair J, Martinez-Becerra FJ, Choudhari SP, Clements JD, Picking WL, Pasetti MF (2013) Evaluation of immunogenicity and protective efficacy of orally delivered Shigella type III secretion system proteins IpaB and IpaD. Vaccine 31:2919–2929CrossRefGoogle Scholar
  30. 30.
    Holmgren J, Lönnroth I, Månsson J, Svennerholm L (1975) Interaction of cholera toxin and membrane GM1 ganglioside of small intestine. Proc Natl Acad Sci USA 72:2520–2524CrossRefGoogle Scholar
  31. 31.
    Ahmadi A, Honari H, Minaei M (2014) Cloning and expression of fusion genes of domain A-1 protective antigen of Bacillus anthracis and Shigella enterotoxin B Subunit (Stxb) in E. coil. J Shahid Sadoughi Univ Med Sci 22Google Scholar
  32. 32.
    Demurtas OC, Massa S, Illiano E, De Martinis D, Chan PK, Di Bonito P, Franconi R (2016) Antigen production in plant to tackle infectious diseases flare up: the case of SARS. Front Plant Sci 7:54CrossRefGoogle Scholar
  33. 33.
    Abboud N, Casadevall A (2008) Immunogenicity of Bacillus anthracis protective antigen domains and efficacy of elicited antibody responses depend on host genetic background. Clin Vaccine Immunol CVI 15:1115–1123CrossRefGoogle Scholar
  34. 34.
    Li Q, Peachman KK, Sower L, Leppla SH, Shivachandra SB, Matyas GR, Peterson JW, Alving CR, Rao M, Rao VB (2009) Anthrax LFn-PA hybrid antigens: biochemistry, immunogenicity, and protection against lethal ames spore challenge in rabbits. Open Vaccine J 2:92–99CrossRefGoogle Scholar
  35. 35.
    Iyer V, Hu L, Schanté CE, Vance D, Chadwick C, Jain NK, Brey RN, Joshi SB, Volkin DB, Andra KK, Bann JG, Mantis NJ, Middaugh CR (2013) Biophysical characterization and immunization studies of dominant negative inhibitor (DNI), a candidate anthrax toxin subunit vaccine. Hum Vaccine Immunother 9:2362–2370CrossRefGoogle Scholar
  36. 36.
    Reason D, Ullal A, Liberato J, Sun J, Keitel W, Zhou J (2008) Domain specificity of the human antibody response to Bacillus anthracis protective antigen. Vaccine 26:4041–4047CrossRefGoogle Scholar
  37. 37.
    Hammamieh R, Ribot WJ, Abshire TG, Jett M, Ezzell J (2008) Activity of the Bacillus anthracis 20 kDa protective antigen component. BMC Infect Dis 8:124CrossRefGoogle Scholar
  38. 38.
    Pogrebnyak N, Golovkin M, Andrianov V, Spitsin S, Smirnov Y, Egolf R, Koprowski H (2005) Severe acute respiratory syndrome (Sars) S protein production in plants: development of recombinant vaccine. Proc Natl Acad Sci Koprowski 102:9062–9067CrossRefGoogle Scholar
  39. 39.
    Chowdhury K, Bagasra O (2007) An edible vaccine for malaria using transgenic tomatoes of varying sizes, shapes and colors to carry different antigens. Med Hypotheses 68:22–30CrossRefGoogle Scholar
  40. 40.
    Sala F, Rigano MM, Barbante A, Basso B, Walmsley AM, Castiglione S (2003) Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine 21:803–808CrossRefGoogle Scholar
  41. 41.
    Jafari D, Dehghan NF (2018) Isolation and bioinformatics study of TbJAMYC transcription factor involved in biosynthesis of taxol from Iranian yew. Rangel For Plant Breed Genet Res 26:12–22Google Scholar
  42. 42.
    Jafari D, Dehghan Nayeri F, Honari H, Hoseini R, Jafari R (2016) Bioinformatic analysis of different fusions of ipaD, PA20 and CTxB antigens: a preliminary analysis for vaccine design. Genet 3rd Millenn 14:4234–4241Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Jafari Davod
    • 1
    • 2
  • Dehghan Nayeri Fatemeh
    • 2
    Email author
  • Hossein Honari
    • 3
  • Ramin Hosseini
    • 2
  1. 1.Medical Biotechnology Department, School of Allied MedicineIran University of Medical SciencesTehranIran
  2. 2.Biotechnology Department, Faculty of Agricultural and Natural SciencesImam Khomeini International University (IKIU)QazvinIran
  3. 3.Faculty of Basic ScienceImam Hussein UniversityTehranIran

Personalised recommendations