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