Anti-VEGFR2 nanobody expression in lettuce using an infectious Turnip mosaic virus vector

  • Malihe Mirzaee
  • Mokhtar Jalali-Javaran
  • Ahmad Moieni
  • Sirous Zeinali
  • Mahdi Behdani
  • Masoud Shams-Bakhsh
  • Mostafa Modarresi
Original Article


Angiogenesis plays an important role in tumor growth and metastasis of cancer. Vascular endothelial growth factor is the key regulator in stimulating angiogenesis. The VEGF activity is mediated by binding to its cell-surface receptors, mainly VEGFR2. Therefore, inhibition of the VEGF/VEGFR2 interaction by antibodies is investigated as a therapeutic strategy in cancer therapy. Here, we describe transient expression of an anti-VEGFR2 nanobody (3VGR19) by a viral vector based on Turnip mosaic virus in lettuce (Lactuca sativa L.). RT-PCR analysis demonstrated the 3VGR19 transcript expression. Western blot analysis showed the 3VGR19 protein expression with an expected molecular mass of ~15 kDa and based on the ELISA results, the expression level of 3VGR19 was 8 μg/g of leaf fresh weight. Taken together, recombinant 3VGR19 could be efficiently expressed in lettuce leaves and TuMV-based expression system would be an appropriate platform for production of recombinant proteins in lettuce plants.


Angiogenesis Lettuce Nanobody Transient expression TuMV 



Vascular endothelial growth factor receptor 2


Turnip mosaic virus


Cauliflower mosaic virus


Green fluorescent protein



The authors gratefully thank Prof. Shyi-Dong Yeh for kindly providing the TuMV-based expression vector p35STuMVGFPHis. We acknowledge the skilled technical assistance of Dr. S. M. Nassaj-Hosseini.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abdoli-Nasab M, Jalali-Javaran M, Cusidό RM, Palazόn J, Baghizadeh A, Alizadeh H (2013) Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast. Mol Biol Rep 40:5749–5758. doi: 10.1007/s11033-013-2678-0 CrossRefPubMedGoogle Scholar
  2. Ahmad P, Ashraf M, Younis M, Hu X, Kumar A, Akram NA, Al-Qurainy F (2012) Role of transgenic plants in agriculture and biopharming. Biotechnol Adv 30:524–540. doi: 10.1016/j.biotechadv.2011.09.006 CrossRefPubMedGoogle Scholar
  3. Ahmadvand D, Rahbarizadeh F, Iri-Sofla FJ, Namazi G, Khaleghi S, Geramizadeh B, Pasalar P, Karimi H, Bakhtiari SHA (2010) Inhibition of angiogenesis by recombinant VEGF receptor fragments. Lab Med 41:417–422. doi: 10.1309/LMMH2WYRLP7B3HJN CrossRefGoogle Scholar
  4. Backer MV, Backer JM (2001) Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of shiga-like toxin-VEGF fusion proteins. Bioconjug Chem 12:1066–1073. doi: 10.1021/bc015534j CrossRefPubMedGoogle Scholar
  5. Basaran P, Rodriguez-Cerezo E (2008) Plant molecular farming: opportunities and challenges. Crit Rev Biotechnol 28:153–172. doi: 10.1080/07388550802046624 CrossRefPubMedGoogle Scholar
  6. Beauchemin C, Bougie V, Laliberté JF (2005) Simultaneous production of two foreign proteins from a potyvirus-based vector. Virus Res 112:1–8. doi: 10.1016/j.virusres.2005.03.001 CrossRefPubMedGoogle Scholar
  7. Behdani M, Zeinali S, Khanahmad H, Karimipour M, Asadzadeh N, Azadmanesh K, Khabiri A, Schoonooghe S, Anbouhi MH, Hassanzageh-Ghassabeh G, Muyldermans S (2011) Generation and characterization of a functional nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Mol Immunol 50:35–41. doi: 10.1016/j.molimm.2011.11.013 CrossRefPubMedGoogle Scholar
  8. Bellou S, Pentheroudakis G, Murphy C, Fotsis T (2013) Anti-angiogenesis in cancer therapy: Hercules and hydra. Cancer Lett 338:219–228. doi: 10.1016/j.canlet.2013.05.015 CrossRefPubMedGoogle Scholar
  9. Boehm R (2007) Bioproduction of therapeutic proteins in the 21st century and the role of plants and plant cells as production platforms. Ann N Y Acad Sci 1102:121–134. doi: 10.1196/annals.1408.009 CrossRefPubMedGoogle Scholar
  10. 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. doi: 10.1016/0003-2697(76)90527-3 CrossRefPubMedGoogle Scholar
  11. Chen CC, Chen TC, Raja JAJ, Chang CA, Chen LW, Lin SS, Yeh SD (2007) Effectiveness and stability of heterologous proteins expressed in plants by Turnip mosaic virus vector at five different insertion sites. Virus Res 130:210–227. doi: 10.1016/j.virusres.2007.06.014 CrossRefPubMedGoogle Scholar
  12. Chung HY, Lee HH, Kim KI, Chung HY, Hwang-Bo J, Park JH, Sunter G, Kim JB, Shon DH, Kim W, Chung IS (2011) Expression of a recombinant chimeric protein of hepatitis A virus VP1-Fc using a replicating vector based on Beet curly top virus in tobacco leaves and its immunogenicity in mice. Plant Cell Rep 30:1513–1521. doi: 10.1007/s00299-011-1062-6 CrossRefPubMedGoogle Scholar
  13. Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, Baetselier PD, Muyldermans S, Revets H (2004) Efficient cancer therapy with a nanobody-based conjugate. Cancer Res 64:2853–2857. doi: 10.1158/0008-5472.CAN-03-3935 CrossRefPubMedGoogle Scholar
  14. Ding G, Chen X, Zhu J, Feng Z (2013) A murine-human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro. Cytotechnology 66:395–411. doi: 10.1007/s10616-013-9587-x CrossRefPubMedPubMedCentralGoogle Scholar
  15. Doran PM (2000) Foreign protein production in plant tissue cultures. Curr Opin Biotechnol 11:199–204. doi: 10.1016/S0958-1669(00)00086-0 CrossRefPubMedGoogle Scholar
  16. Farajpour Z, Rahbarizadeh F, Kazemi B, Ahmadvand D (2014) Nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment. Biochem Biophys Res Commun 446:132–136. doi: 10.1016/j.bbrc.2014.02.069 CrossRefPubMedGoogle Scholar
  17. Fischer R, Twyman RM, Schillberg S (2003) Production of antibodies in plants and their use for global health. Vaccine 21:820–825. doi: 10.1016/S0264-410X(02)00607-2 CrossRefPubMedGoogle Scholar
  18. Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158. doi: 10.1016/j.pbi.2004.01.007 CrossRefPubMedGoogle Scholar
  19. Fleetwood F, Klint S, Hanze M, Gunneriusson E, Frejd FY, Ståhl S, Löfblom J (2014) Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity. Sci Rep 4:7518. doi: 10.1038/srep07518 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Folkman J (2005) Antiangiogenesis in cancer therapy—endostatin and its mechanisms of action. Exp Cell Res 312:594–607. doi: 10.1016/j.yexcr.2005.11.015 CrossRefPubMedGoogle Scholar
  21. Gleba Y, Marillonnet S, Klimyuk V (2004) Engineering viral expression vectors for plants: the ‘full virus’ and the ‘deconstructed virus’ strategies. Curr Opin Plant Biol 7:182–188. doi: 10.1016/j.pbi.2004.01.003 CrossRefPubMedGoogle Scholar
  22. Gleba Y, Klimyuk V, Marillonnet S (2005) Magnifectiion—a new platform for expressing recombinant vaccines in plants viral expression vectors for plants. Vaccine 23:2041–2048. doi: 10.1016/j.vaccine.2005.01.006 CrossRefGoogle Scholar
  23. Hendy S, Chen ZC, Barker H, Cruz SS, Chapman S, Torrance L, Cockburn W, Whitelam GC (1999) Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector. J Immunol Methods 231:137–146. doi: 10.1016/S0022-1759(99)00150-7 CrossRefPubMedGoogle Scholar
  24. Hernot S, Unnikrishnan S, Du Z, Shevchenko T, Cosyns B, Broisat A, Toczek J, Caveliers V, Muyldermans S, Lahoutte T, Klibanov AL, Devoogdt N (2011) Nanobody-coupled microbubbles as novel molecular tracer. J Control Release 158:346–353. doi: 10.1016/j.jconrel.2011.12.007 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Holmes K, Roberts OL, Thomas AM, Cross MJ (2007) Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition. Cell Signal 19:2003–2012. doi: 10.1016/j.cellsig.2007.05.013 CrossRefPubMedGoogle Scholar
  26. Huang Z, Le Pore K, Elikin G, Thanavala Y, Mason HS (2007) High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system. Plant Biotechnol J 6:202–209. doi: 10.1111/j.1467-7652.2007.00316.x CrossRefGoogle Scholar
  27. Joh LD, Wroblewski T, Ewing NN, VanderGheynst JS (2005) High-level transient expression of recombinant protein in lettuce. Biotechnol Bioeng 91:861–871. doi: 10.1002/bit.20557 CrossRefPubMedGoogle Scholar
  28. Karasev AV, Foulke S, Wellens C, Rich A, Shon KJ, Zwierzynski I, Hone D, Koprowski H, Reitz M (2004) Plant based HIV-1 vaccine candidate: tat protein produced in spinach. Vaccine 23:1875–1880. doi: 10.1016/j.vaccine.2004.11.021 CrossRefGoogle Scholar
  29. Kazemi-Lomedasht F, Behdani M, Bagheri KP, Habibi-Anbouhi M, Abolhassani M, Arezumand R, Shahbazzadeh D, Mirzahoseini H (2015) Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody. Mol Immunol 65:58–67. doi: 10.1016/j.molimm.2015.01.010 CrossRefPubMedGoogle Scholar
  30. Kim KI, Chung HY, Yoo KH, Park JH, Lee HH, Soek YJ, Ko KS, Kang HS, Lee KJ, Oh DB, Joung YH, Chung IS (2012) Expression of a recombinant chimeric protein of human colorectal cancer antigen GA733-2 and Fc fragment of antibody using a replicating vector based on Beet curly top virus in infiltrated Nicotiana benthamiana leaves. Plant Biotechnol Rep 6:233–242. doi: 10.1007/s11816-012-0218-3 CrossRefGoogle Scholar
  31. Kolkman JA, Law DA (2010) Nanobodies—from llamas to therapeutic proteins. Drug Discov Today Technol 7:139–146. doi: 10.1016/j.ddtec.2010.03.002 CrossRefGoogle Scholar
  32. Lai H, He J, Engle M, Diamond MS, Chen Q (2011) Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce. Plant Biotechnol J 10:95–104. doi: 10.1111/j.1467-7652.2011.00649.x CrossRefPubMedPubMedCentralGoogle Scholar
  33. Li J, Chen M, Liu XW, Zhang HC, Shen FF, Wang GP (2006) Transient expression of an active human interferon-beta in lettuce. Sci Hortic 112:258–265. doi: 10.1016/j.scienta.2006.12.047 CrossRefGoogle Scholar
  34. Lombardi R, Circelli P, Villani ME, Buriani G, Nardi L, Coppola V, Bianco L, Benvenuto E, Donini M, Marusic C (2009) High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol 96:1–11. doi: 10.1186/1472-6750-9-96 Google Scholar
  35. Love AJ, Chapman SN, Matic S, Noris E, Lomonossoff GP, Taliansky M (2012) In planta production of a candidate vaccine against bovine papillomavirus type 1. Planta 236:1305–1313. doi: 10.1007/s00425-012-1692-0 CrossRefPubMedGoogle Scholar
  36. Matsui T, Asao H, Ki M, Sawada K, Kato K (2009) Transgenic lettuce producing a candidate protein for vaccine against edema disease. Biosci Biotechnol Biochem 73:1628–1634. doi: 10.1271/bbb.90129 CrossRefPubMedGoogle Scholar
  37. Muyldermans S (2001) Single domain camel antibodies: current status. Rev Mol Biotechnol 74:277–302. doi: 10.1016/S1389-0352(01)00021-6 CrossRefGoogle Scholar
  38. Negrouk V, Eisner G, Lee H, Han K, Tavlor D, Wong HC (2005) Highly efficient transient expression of functional recombinant antibodies in lettuce. Plant Sci 169:433–438. doi: 10.1016/j.plantsci.2005.03.031 CrossRefGoogle Scholar
  39. Ohshima K, Tanaka M, Sako N (1996) The complete nucleotide sequence of turnip mosaic virus RNA Japanese strain. Arch Virol 141:1991–1997. doi: 10.1007/BF01718209 CrossRefPubMedGoogle Scholar
  40. Oliveira S, Heukers R, Sornkom J, Kok RJ, van Bergen En Henegouwen PMP (2013) Targeting tumors with nanobodies for cancer imaging and therapy. J Control Release 172:607–617. doi: 10.1016/j.jconrel.2013.08.298 CrossRefPubMedGoogle Scholar
  41. Omidfar K, Zanjani FSA, Hagh AG, Azizi MD, Rasouli SJ, Kashanian S (2013) Efficient growth inhibition of EGFR over-expressing tumor cells by an anti-EGFR nanobody. Mol Biol Rep 40:6737–6745. doi: 10.1007/s11033-013-2790-1 CrossRefPubMedGoogle Scholar
  42. Ritala A, Häkkinen T, Schillberg S (2014) Molecular pharming in plants and plant cell cultures: a great future ahead? Pharm Bioprocess 2:223–226. doi: 10.4155/pbp.14.21 CrossRefGoogle Scholar
  43. Roggero P, Ciuffo M, Benvenuto E, Franconi R (2001) The expression of a single-chain Fv antibody fragment in different plant hosts and tissues by using Potato Virus X as a vector. Protein Expr Purif 22:70–74. doi: 10.1006/prep.2001.1398 CrossRefPubMedGoogle Scholar
  44. Sabalza M, Christou P, Capell T (2014) Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnol Lett 12:2367–2379. doi: 10.1007/s10529-014-1621-3 CrossRefGoogle Scholar
  45. Saerens D, Ghassabeh GH, Muyldermans S (2008) Single-domain antibodies as building blocks for novel therapeutics. Curr Opin Pharmacol 8:600–608. doi: 10.1016/j.coph.2008.07.006 CrossRefPubMedGoogle Scholar
  46. Scott A, Mellor H (2009) VEGF receptor trafficking in angiogenesis. Biochem Soc Trans 37:1184–1188. doi: 10.1042/BST0371184 CrossRefPubMedGoogle Scholar
  47. Shahangian SS, Sajedi RH, Hasannia S, Jalili S, Mohammadi M, Taghdir M, Shali A, Mansouri K, Sariri R (2015) A conformation-based phage-display panning to screen neutralizinganti-VEGF VHHs with VEGFR2 mimicry behavior. Int J Biol Macromol 77:222–234. doi: 10.1016/j.ijbiomac.2015.02.047 CrossRefPubMedGoogle Scholar
  48. Shih SMH, Doran PM (2009) Foreign protein production using plant cell and organ cultures: advantages and limitations. Biotechnol Adv 27:1036–1042. doi: 10.1016/j.biotechadv.2009.05.009 CrossRefPubMedGoogle Scholar
  49. Shojaei F (2012) Anti-angiogenesis therapy in cancer: current challenges and future perspectives. Cancer Lett 320:130–137. doi: 10.1016/j.canlet.2012.03.008 CrossRefPubMedGoogle Scholar
  50. Song L, Zhao DG, Wu YJ, Li Y (2008) Transient expression of chicken alpha interferon gene in lettuce. J Zhejiang Univ Sci B 9:351–355. doi: 10.1631/jzus.B0710596 PubMedPubMedCentralGoogle Scholar
  51. Stoger E, Sack M, Fischer R, Christou P (2002) Plantibodies: applications, advantages and bottlenecks. Curr Opin Biotechnol 13:161–166. doi: 10.1016/S0958-1669(02)00303-8 CrossRefPubMedGoogle Scholar
  52. Teh YHA, Kavanagh TA (2010) High-level expression of Camelid nanobodies in Nicotiana benthamiana. Transgenic Res 19:575–586. doi: 10.1007/s11248-009-9338-0 CrossRefPubMedGoogle Scholar
  53. Tourino A, Sánchez F, Fereres A, Ponz F (2008) High expression of foreign proteins from a biosafe viral vector derived from Turnip mosaic virus. Span J Agric Res 6:48–58. doi: 10.5424/sjar/200806S1-373 CrossRefGoogle Scholar
  54. Twyman RM, Stoger E, Schillberg S, Christou P, Fischer R (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21:570–578. doi: 10.1016/j.tibtech.2003.10.002 CrossRefPubMedGoogle Scholar
  55. Vaneycken I, Govaert J, Vincke C, Caveliers V, Lahoutte T, Baetselier PD, Raes G, Bossuyt A, Muyldermans S, Devoogdt N (2010) In vitro analysis and in vivo tumor targeting of a humanized, grafted nanobody in mice using pinhole SPECT/micro-CT. J Nucl Med 51:1099–1106. doi: 10.2967/jnumed.109.069823 CrossRefPubMedGoogle Scholar
  56. Vardakou M, Sainsbury F, Rigby N, Mulholland F, Lomonossoff GP (2012) Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system. Protein Expr Purif 81:69–74. doi: 10.1016/j.pep.2011.09.005 CrossRefPubMedGoogle Scholar
  57. Wada S, Tsunoda T, Baba T, Primus FJ, Kuwano H, Shibuya M, Tahara H (2005) Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2. Cancer Res 65:4939–4946. doi: 10.1158/0008-5472.CAN-04-3759 CrossRefPubMedGoogle Scholar
  58. Wagner B, Fuchs H, Adhami F, Ma Y, Scheiner O, Breiteneder H (2003) Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana. Methods 32:227–234. doi: 10.1016/j.ymeth.2003.08.005 CrossRefGoogle Scholar
  59. Zelada AM, Calamante G, de la Paz Santangelo M, Bigi F, Verna F, Mentaberry A, Cataldi A (2006) Expression of tuberculosis antigen ESAT-6 in Nicotiana tabacum using a potato virus X-based vector. Tuberculosis 86:263–267. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  60. Zhang W, Ran S, Sambade M, Huang X, Thorpe PE (2002) A monoclonal antibody that blocks VEGF binding to VEGFR2 (KDR/Flk-1) inhibits vascular expression of Flk-1 and tumor growth in an orthotopic human breast cancer model. Angiogenesis 5:34–44. doi: 10.1023/A:1021540120521 CrossRefGoogle Scholar
  61. Zhang Z, Neiva KG, Lingen MW, Ellis LM, Nör JE (2009) VEGF-dependent tumor angiogenesis requires inverse and reciprocal regulation of VEGFR1 and VEGFR2. Cell Death Differ 17:499–512. doi: 10.1038/cdd.2009.152 CrossRefPubMedPubMedCentralGoogle Scholar
  62. Zhao MA, An SJ, Lee SC, Kim DS, Kang BC (2013) Overexpression of a single-chain cariable fragment (scFv) antibody confers unstable resistance to TuMV in Chinese cabbage. Plant Mol Biol Rep 31:1203–1211. doi: 10.1007/s11105-013-0577-0 CrossRefGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2017

Authors and Affiliations

  • Malihe Mirzaee
    • 1
  • Mokhtar Jalali-Javaran
    • 1
  • Ahmad Moieni
    • 1
  • Sirous Zeinali
    • 2
  • Mahdi Behdani
    • 3
  • Masoud Shams-Bakhsh
    • 4
  • Mostafa Modarresi
    • 1
  1. 1.Department of Plant Breeding and Biotechnology, Faculty of AgricultureTarbiat Modares UniversityTehranIran
  2. 2.Department of Molecular MedicinePasteur Institute of IranTehranIran
  3. 3.Venom and Biotherapeutics Molecules Lab., Biotechnology Department, Biotechnology Research CenterPasteur Institute of IranTehranIran
  4. 4.Department of Plant Pathology, Faculty of AgricultureTarbiat Modares UniversityTehranIran

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