NICOTIANA TABACUM AS A POTENTIAL PLATFORM FOR THE PRODUCTION OF RECOMBINANT ANTI-TOXOPLASMA SINGLE-CHAIN FRAGMENT (scFv) ANTIBODY
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Published:
Jun 30, 2020
Keywords:
Plant transformation plant molecular farming recombinant protein single-chain variable fragment antibody (scFv) tobacco toxoplasmosis
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Abstract
Abstract
Plant systems have now gained much attention as they provide low risk of pathogen contamination and cost of production, appropriate post-translational modification, and multimeric assembly capability compared to the mammalian and bacterial hosts. In this study, we aimed to produce single-chain fragment antibody (scFv) encoding anti-Toxoplasma proteins, TP60 in Nicotiana tabacum cv. SR1. Leaf explants of N. tabacum were infected with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pCAMBIA1304 containing TP60 gene. Putative transformants were confirmed through GUS and GFP qualitative assays. Bands observed at the predicted size of 914 bp confirmed the presence of TP60 transgene and the transgene was stably integrated in both T0 and T1 tobacco genome. FV12-6 transgenic line produced the highest mRNA expression (7-fold) correlated to the highest accumulation of anti-Toxoplasma recombinant scFv antibody (0.52 % of the total soluble protein), followed by FV16-10 (0.25 %), FV17-7 and FV3-11 (0.18 %). The expression of TP60 transgene did not affect the growth of the transgenic plants and the segregation analysis of FV3, FV12, and FV16 in T1 generation confirmed the transgene integration within a single locus according to 3:1 Mendelian’s law. These findings indicated the possibility of using plants as a bio-factory for recombinant protein production.
Abstrak
Sistem tumbuhan kini telah mendapat banyak perhatian kerana mereka menyediakan risiko yang rendah terhadap pencemaran patogen dan kos pengeluaran, pengubahsuaian pasca-protein translasi yang sesuai, dan keupayaan penggabungan multimerik berbanding dengan tentera mamalia dan bakteria. Dalam kajian ini, kami berhasrat untuk menghasilkan antibodi serasi rantaian tunggal (scFv) yang mengekodkan protein anti-Toxoplasma, TP60 di Nicotiana tabacum cv. SR1. Eksplan daun N. tabacum telah dijangkiti oleh Agrobacterium tumefaciens strain LBA4404 yang menyimpan vektor dedua pCAMBIA1304 yang mengandungi gen TP60. Transforman putative disahkan melalui ujian kualitatif GUS dan GFP. Band-band yang diperhatikan pada saiz ramalan 914 bp mengesahkan kehadiran TP60 transgen dan transgen tersebut telah diintegrasi secara stabil dalam genom tembakau T0 dan T1. Individu transgenik FV12-6 menghasilkan ekspresi mRNA tertinggi (7 kali ganda) berkait dengan pengumpulan tertinggi antibodi rekombinan scFv anti-Toxoplasma (0.52% daripada jumlah protein larut), diikuti oleh FV16-10 (0.25%), FV17-7 dan FV3-11 (0.18%). Ungkapan transgenik TP60 tidak menjejaskan pertumbuhan tumbuhan transgenik dan analisis pemisahan FV3, FV12, dan FV16 dalam generasi T1 mengesahkan integrasi transgene dalam satu lokus mengikut 3: 1 undang-undang Mendelian. Penemuan ini menunjukkan kemungkinan menggunakan tumbuhan sebagai kilang bio untuk pengeluaran protein rekombinan.
Keywords
Agrobacterium-mediated transformation, plant molecular farming, recombinant protein, single-chain fragment antibody (scFv), tobacco, toxoplasmosis
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Mohamad Zoolkefli, F. I. R., Go, P. S., Tan, B. C., Rejab, N. A., Othman, R. Y., & Khalid, N. (2020). NICOTIANA TABACUM AS A POTENTIAL PLATFORM FOR THE PRODUCTION OF RECOMBINANT ANTI-TOXOPLASMA SINGLE-CHAIN FRAGMENT (scFv) ANTIBODY. Malaysian Journal of Science, 39(2), 1–26. https://doi.org/10.22452/mjs.vol39no2.1
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Licensee MJS, Universiti Malaya, Malaysia. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
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Chen GQ. (2011). Effective reduction of chimeric tissue in transgenics for the stable genetic transformation of Lesquerella fendleri. HortScience 46(1): 86-90.
Chen Q. (2015). Plant-made vaccines against West Nile virus are potent, safe, and economically feasible. Biotechnology Journal 10(5): 671-680.
Cummings JF., Guerrero ML., Moon JE., Waterman P., Nielsen RK., Jefferson S., Gross FL., Hancock K., Katz JM. & Yusibov, V. (2014). Safety and immunogenicity of a plant-produced recombinant monomer hemagglutinin-based influenza vaccine described from influenza A (H1N1) pdm09 virus: A Phase 1 dose-escalation study in healthy adults. Vaccines 32(19): 2251-2259.
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Dobhal S., Chaudhary VK., Singh A., Pandey D., Kumar A. & Agrawal, S. (2013). Expression of recombinant antibody (single chain antibody fragment) in transgenic plant Nicotiana tabacum cv. Xanthi. Molecular Biology Report 40(12): 7027-7037.
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Eluk D., Nagel OG., Zimmerman J., Molina MP. & Althaus, RL. (2016). Effect of antibiotics on the germination and root elongation of argentine intensive crops. International Journal of Environmental Research 10(4): 471-480.
Farajnia S., Ahmadzadeh V., Tanomand A., Veisi K., Khosroshahi SA. & Rahbarnia, L. (2014). Development trends for generation of single-chain antibody fragments. Immunopharmacology and Immunotoxicology (36)5: 297-308.
Flego M., Frau A., Accardi L.,Mallano A., Ascione A., Gellini M., Fanunza E., Vella S., Bonito PD. & Tramontano, E. (2019). Intracellular human antibody fragments recognizing the VP35 protein of Zaire Ebola filovirus inhibit the protein activity. BMC Biotechnology 19(64).
Flegr J., Prandota J., Sovičková M. & Israili, ZH. (2014). Toxoplasmosis – A Global Threat. Correlation of Latent Toxoplasmosis with Specific Disease
Burden in a Set of 88 Countries. PLOS ONE 9(3): e90203.
Foroutan M., Ghaffarifar F., Sharifi Z., Dalimi A. & Jorjani, O. (2019). Rhoptry antigens as Toxoplasma gondii vaccine target. Clinical and Experimental Vaccine Research. 8(1): 4-26.
Giersberg M., Floss DM., Kipriyanov S., Conrad U. & Scheller, J. (2010). Covalent dimerization of camelidae anti-human TNF-alpha single domain antibodies by the constant kappa light chain domain improves neutralizing activity. Biotechnology and Bioengineering 106(1): 161-166.
Go PS. (2013). Expression of anti-Toxoplasma scFv antibodies in plants. Dissertation, University of Malaya.
Goldman JJ., Hanna WW., Fleming G. & Ozias-Akins, P. (2003). Fertile transgenic pearl millet [Pennisetum glaucum (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues. Plant Cell Reports 21: 482-486.
Innes EA., Bartley PM., Rocchi M., Benavidas-Silvan J., Burrells A., Hotchkiss E., Chianini F., Canton G. & Katzer, F. (2011). Developing vaccines to control protozoan parasites in ruminants: dead or alive? Veterinary Parasitology 180(1-2): 155–163.
Jefferson RA., Kavanagh TA. & Bevan, MW. (1987). GUS Fusions: β-glucuronidase as a sensitive and versatile gene marker in higher plants. EMBO Journal 6: 3901-3907.
Kamaté K., Rodriguez-Llorente ID., Scholte M., Durand P., Ratet P., Kondorosi A. & Trinh, TH. (2000). Transformation of floral organs with GFP in Medicago truncatula. Plant Cell Reports 19: 647-653.
Katja MA., Kristian MM. & Andreas, P. (1998). Factors Influencing the Dimer to Monomer Transition of an Antibody Single-Chain Fv Fragment. Biochemistry 37(37): 12918-12926.
Kazan K. & Lyons, R. (2016). The link between flowering time and stress tolerance. Journal of Experimental Botany 67(1): 47–60.
Kemski MM., Stevens B. & Rappleye, CA. (2013). Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum. Fungal Biology 117(1): 41-51.
Kim MY., Reljic R., Kilbourne J., Ceballos-Olvera I., Yang MS., Reyes-del Valle J. & Maso, HS. (2015). Novel vaccination approach for dengue infection based on recombinant immune complex universal platform. Vaccine 33(15): 1830-1838.
Kurasawa JH., Shestopal SA., Jha NK., Ovanesov MV., Lee TK. & Sarafanov, AG. (2012). Insect cell-based expression and characterization of a single-chain variable antibody fragment directed against blood coagulation factor VIII. Protein Expression and Purification 88(2): 201-206.
Lai H., He J., Hurtado J., Stahnke J., Fuchs A., Mehlhop E., Gorlatov S., Loos A., Diamond MS. & Chen, Q. (2014).
Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants. Plant Biotechnology Journal 12(8): 1098-1107.
Lim SSY. (2012). The development of single-chain variable fragment (scFv) antibodies against Toxoplasma gondii by phage-display, Dissertation, University of Malaya.
Lim SSY., Chua KH., Nölke G., Spiegel H., Goh WL., Chow SK., Kee BP., Fisher R., Schillberg S. & Othman, RY. (2018). Plant-derived chimeric antibodies inhibit the invasion of human fibroblasts by Toxoplasma gondii. PeerJ 6(e5780).
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