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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References
Ahmad ZA., Yeap SK., Ali AM., Ho WY., Alitheen NBM. & Hamid, M. (2012a). scFv antibody: principles and clinical application. Clinical and Development Immunology 2012: 1-15.
Ahmad P., Ashraf M., Younis M., Hu X., Kumar A., Akram NA. & Al-Qurany, F. (2012b). Role of transgenic plants in agriculture and biopharming. Biotechnology Advances 30(3): 524-540.
Amaro MO., Xisto MF., Dias AS., Versiani AF., Cardoso SA., Otoni WC., da Silva CC. & De Paula, SO. (2015). Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes. Plant Cell Reports 34(6): 919-928.
Artimo P., Jonnalagedda M., Arnold K., Baratin D., Csardi G., de Castro E., Duvaud S., Flegel V., Fortier A., Gasteiger E., Grosdidier A., Hernandez C., Ioannidis V., Kuznetsov D., Liechti R., Moretti S., Mostaguir K., Redaschi N., Rossier G.,
Xenarios I. & Stockinger, H. (2012). ExPASy: SIB bioinformatics resource portal. Nucleic Acids Research 40(W1): W597-W603.
Bradford, MM. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 7(72): 248-254.
Burrells A., Benavides J., Cantón G., Garcia JL., Bartley PM., Nath M., Thomson J., Chianini F., Innes EA. & Katzer, F. (2015). Vaccination of pigs with the S48 strain of Toxoplasma gondii-safer meat for human consumption. Veterinary Research 46(47).
Bustin SA., Benes V., Garson JA., Hellemans J., Hugget J., Kubista M., Mueller R., Nolan T., Pfaffl MW., Shipley GL., Vandesompele J. & Wittwer, CT. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55(4): 611-622.
Buxton D. & Innes, EA. (1995). A commercial vaccine for ovine toxoplasmosis. Parasitology 110: S11-S116.
Buxton D. & Rodger, S.M. (2008). Toxoplasmosis and neosporosis, pp. 112-118, New Jersey, USA: Wiley-Blackwell, Hoboken.
Cérutti M. & Golay, J. (2012). Lepidopteran cells, an alternative for the production of recombinant antibodies? mAbs 4(3): 294–309.
Chaudhry SA., Gad N. & Koren, G. (2014). Toxoplasmosis and pregnancy. Canadian family physician. Medecin de Famille Canadien 60(4): 334–336.
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.
Datta K., Tu J., Oliva N., Ona I., Velazhahan R., Mew TW., Muthukrishnan S. & Datta, SK. (2001). Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars. Plant Science 160(3): 405-414.
Dent M., Hurtado J., Paul AM., Sun H., Lai H., Yang M., Esqueda A., Bai F., Steinkellner H. & Chen, Q. (2016). Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity. Journal of General Virology 97(12): 3280-3290.
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.
Elfahal AM., Elhassan AM., Hussien MO., Enan KA., Musa AB. & El Hussein, AM. (2013). Seroprevalence of Toxoplasma gondii in Dairy Cattle with Reproductive Problems in Sudan. ISRN veterinary science 2013(895165).
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).
Lim SSY. & Othman, RY. (2014). Recent Advances in Toxoplasma gondii Immunotherapeutics. Korean Journal of Parasitology 52(6): 581-593.
Livak KJ. & Schmittgen, TD. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25: 402-408.
Makvandi-Nejad S., McLean MD., Hirama T., Almquist KC., Mackenzie CR. & Hall, JC. (2005). Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B. Transgenic Research 14(5): 785-792.
Maxmen A. (2012). Drug making plant blooms: Approval of a ‘biologic’ manufactured in plant cells may pave the way for similar products. Nature Biotechnology 485: 160.
Merlin M., Gecchele E., Capaldi S., Pezzotti M. & Avesani, L. (2014). Comparative evaluation of recombinant protein production in different biofactories: The green perspective. BioMed Research International 2014(136419).
Murashige T. & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Journal of Plant Physiology 15: 473-497.
Niemer M., Mehofer U., Acosta JAT., Verdianz M., Henkel T., Loos A., Strasser R.,
Maresch D., Rademacher T., Steinkellner H. & Mach, L. (2014). The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: Down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms. Biotechnology Journal 9: 493-500.
Obeme OO., Popoola JO., Leelavathi S. & Reddy, SV. (2011). Advances in plant molecular farming. Biotechnology Advances 29: 210-222.
Omar MTC. (2017). Expression ofFunctional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells. Advanced Pharmaceutical Bulletin 7(2): 299-312.
Puetz J. & Wurm, FM. (2019). Recombinant Proteins for Industrial versus Pharmaceutical Purposes: A Review of Process and Pricing. Processes 7(8): 476-486.
Rahimzadeh M., Sadeghizadeh M., Najafi F., Arab S. & Mobasheri, H. (2016).
Impact of heat shock step on bacterial transformation efficiency. Molecular Biology Research Communications 5(4): 257-261.
Robert S., Khalf M., Goulet MC., D’Aoust MA., Sainsbury F. & Michaud, D. (2013). Protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of Nicotiana benthamiana. Plos One 8(7): 1-9.
Sack M., Hofbauer A., Fischer R. & Stoger, E. (2015). The increasing value of plant-made proteins. Current Opinion in Biotechnology 32: 163-170.
Sarker A., Rathore AS. & Gupta, RD. (2019). Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process. Microbial Cell Factories 18(5).
Schmidt GW. & Delaney, SK. (2010). Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Molecular Genetics and Genomics 283(3): 233-241.
Schouten A., Roosien J., Bakker J. & Schots, A. (2002). Formation of disulphide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol. The Journal of Biological Chemistry 277(31): 19339-19345.
Shoji Y., Jones RM., Mett V., Chichester JA., Musiychuk K., Sun X., Tumpey TM., Green BJ., Shamloul M., Norikane J., Bi H., Hartman CE., Bottone C., Stewart M., Streatfield SJ. & Yusibov, V. (2013). A plant-produced H1N1 trimeric
hemagglutinin protects mice from a lethal influenza virus challenge. Human, Vaccines, and Immunotherapeutics 9(3): 553-560.
Stelzer S., Basso W., Silvan JB., Ortega-Mora LM., Maksimov P., Gethman J., Conraths FJ. & Schares, G. (2019). Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact. Food and Waterborne Parasitology 15(e00037).
Takeno K. (2012). Stress-induced flowering. In H Hirt and K Shinozaki (Eds.), Abiotic Stress Responses in Plants (pp.331-345). New York, NY: Springer.
Takeno K. (2016). Stress-induced flowering: the third category of flowering response. Journal of Experimental Botany 67(17): 4925-4934.
Takeyama N., Kiyono H. & Yuki, Y. (2015). Plant-based vaccines for animals and humans: recent advances in technology and clinical trials. Therapeutic Advances in Vaccine 3(5-6): 139-154.
Tamura M., Togami J., Ishiguro K., Nakamura N., Katsumoto Y., Suzuki K., Kusumi T. & Tanaka, Y. (2003). Regeneration of Verbena (Verbena x hybrida) by Agrobacterium tumefaciens. Plant Cell Reports 21(5): 459-466.
Tizaoui K., & Kchouk, ME. (2012). Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco. Genetics and molecular biology 35(3): 640–649.
Wang JL., Zhang NZ., Li TT., He JJ., Elsheikha HM. & Zhu, XQ. (2019). Advances in the development of anti-Toxoplasma gondii vaccines: Challenges, opportunities, and perspectives. Trends Parasitology 35(3): 239-253.
Wang XF., Li L., Yang T., Lui J., Fan Y., Zhu X. & Wang, XZ. (2015). Single-chain variable fragment (scFv) expression in tobacco plants via agroinoculation. Russian Journal of Plant Physiology 62(3): 401–407.
Wang ZD., Liu HH., Ma ZX., Ma HY., Li ZY., Yang ZB., Zhu XQ., Xu B., Wei F. & Liu, Q. (2017). Toxoplasma gondii Infection in Immunocompromised Patients: A Systematic Review and Meta-Analysis. Frontiers in Microbiology 8(389).
Wen-Bin Y., Jin-Lei W., Qian G., Yang Z., Kai C., Qing L., Qin-Li L., Xing-Quan Z. & Dong-Hui Z. (2019). Immunization With a Live-Attenuated RH:ΔNPT1 Strain of Toxoplasma gondii Induces Strong Protective Immunity Against Toxoplasmosis in Mice. Frontiers in Microbiology 10: 1875-1887.
Yao J., Weng Y., Dickey A. & Wang, KY. (2015). Plants as factories for Human Pharmaceuticals: Applications and Challenges. International Journal of Molecular Science 16(12): 28549-28565.
Yuan X., Chen X., Yang M., Hu J., Yang W., Chen T., Wang Q., Zhang X., Lin R. & Zhao, A. (2016). Efficient construct of a large and functional scFv yeast display library derived
from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated
recombination. Applied of Microbiology and Biotechnology 100(9): 4051-4061.
Yusakul G., Sakamoto S., Juengwatanatrakul T., Putalun W., Tanaka H. & Morimoto, S. (2015). Preparation and application of a monoclonal antibody against the isoflavone glycoside daidzin using a mannich reaction‐derived hapten conjugate. Phytochemical Analysis 27(1): 81-88.
Zhou X., Carranco R., Vitha S. & Hall, TC. (2005). The dark side of green fluorescent protein. New Phytologist 168: 313-322.
Zhou X., Chandrasekharan MB. & Hall, TC. (2004). High rooting frequency and functional analysis of GUS and GFP expression in transgenic Medicago truncatula A17. New Phytologist 162: 813-822.
Ahmad P., Ashraf M., Younis M., Hu X., Kumar A., Akram NA. & Al-Qurany, F. (2012b). Role of transgenic plants in agriculture and biopharming. Biotechnology Advances 30(3): 524-540.
Amaro MO., Xisto MF., Dias AS., Versiani AF., Cardoso SA., Otoni WC., da Silva CC. & De Paula, SO. (2015). Antigen production using heterologous expression of dengue virus-2 non-structural protein 1 (NS1) in Nicotiana tabacum (Havana) for immunodiagnostic purposes. Plant Cell Reports 34(6): 919-928.
Artimo P., Jonnalagedda M., Arnold K., Baratin D., Csardi G., de Castro E., Duvaud S., Flegel V., Fortier A., Gasteiger E., Grosdidier A., Hernandez C., Ioannidis V., Kuznetsov D., Liechti R., Moretti S., Mostaguir K., Redaschi N., Rossier G.,
Xenarios I. & Stockinger, H. (2012). ExPASy: SIB bioinformatics resource portal. Nucleic Acids Research 40(W1): W597-W603.
Bradford, MM. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 7(72): 248-254.
Burrells A., Benavides J., Cantón G., Garcia JL., Bartley PM., Nath M., Thomson J., Chianini F., Innes EA. & Katzer, F. (2015). Vaccination of pigs with the S48 strain of Toxoplasma gondii-safer meat for human consumption. Veterinary Research 46(47).
Bustin SA., Benes V., Garson JA., Hellemans J., Hugget J., Kubista M., Mueller R., Nolan T., Pfaffl MW., Shipley GL., Vandesompele J. & Wittwer, CT. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55(4): 611-622.
Buxton D. & Innes, EA. (1995). A commercial vaccine for ovine toxoplasmosis. Parasitology 110: S11-S116.
Buxton D. & Rodger, S.M. (2008). Toxoplasmosis and neosporosis, pp. 112-118, New Jersey, USA: Wiley-Blackwell, Hoboken.
Cérutti M. & Golay, J. (2012). Lepidopteran cells, an alternative for the production of recombinant antibodies? mAbs 4(3): 294–309.
Chaudhry SA., Gad N. & Koren, G. (2014). Toxoplasmosis and pregnancy. Canadian family physician. Medecin de Famille Canadien 60(4): 334–336.
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.
Datta K., Tu J., Oliva N., Ona I., Velazhahan R., Mew TW., Muthukrishnan S. & Datta, SK. (2001). Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars. Plant Science 160(3): 405-414.
Dent M., Hurtado J., Paul AM., Sun H., Lai H., Yang M., Esqueda A., Bai F., Steinkellner H. & Chen, Q. (2016). Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity. Journal of General Virology 97(12): 3280-3290.
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.
Elfahal AM., Elhassan AM., Hussien MO., Enan KA., Musa AB. & El Hussein, AM. (2013). Seroprevalence of Toxoplasma gondii in Dairy Cattle with Reproductive Problems in Sudan. ISRN veterinary science 2013(895165).
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).
Lim SSY. & Othman, RY. (2014). Recent Advances in Toxoplasma gondii Immunotherapeutics. Korean Journal of Parasitology 52(6): 581-593.
Livak KJ. & Schmittgen, TD. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25: 402-408.
Makvandi-Nejad S., McLean MD., Hirama T., Almquist KC., Mackenzie CR. & Hall, JC. (2005). Transgenic tobacco plants expressing a dimeric single-chain variable fragment (scfv) antibody against Salmonella enterica serotype Paratyphi B. Transgenic Research 14(5): 785-792.
Maxmen A. (2012). Drug making plant blooms: Approval of a ‘biologic’ manufactured in plant cells may pave the way for similar products. Nature Biotechnology 485: 160.
Merlin M., Gecchele E., Capaldi S., Pezzotti M. & Avesani, L. (2014). Comparative evaluation of recombinant protein production in different biofactories: The green perspective. BioMed Research International 2014(136419).
Murashige T. & Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Journal of Plant Physiology 15: 473-497.
Niemer M., Mehofer U., Acosta JAT., Verdianz M., Henkel T., Loos A., Strasser R.,
Maresch D., Rademacher T., Steinkellner H. & Mach, L. (2014). The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: Down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms. Biotechnology Journal 9: 493-500.
Obeme OO., Popoola JO., Leelavathi S. & Reddy, SV. (2011). Advances in plant molecular farming. Biotechnology Advances 29: 210-222.
Omar MTC. (2017). Expression ofFunctional Anti-p24 scFv 183-H12-5C in HEK293T and Jurkat T Cells. Advanced Pharmaceutical Bulletin 7(2): 299-312.
Puetz J. & Wurm, FM. (2019). Recombinant Proteins for Industrial versus Pharmaceutical Purposes: A Review of Process and Pricing. Processes 7(8): 476-486.
Rahimzadeh M., Sadeghizadeh M., Najafi F., Arab S. & Mobasheri, H. (2016).
Impact of heat shock step on bacterial transformation efficiency. Molecular Biology Research Communications 5(4): 257-261.
Robert S., Khalf M., Goulet MC., D’Aoust MA., Sainsbury F. & Michaud, D. (2013). Protection of recombinant mammalian antibodies from development-dependent proteolysis in leaves of Nicotiana benthamiana. Plos One 8(7): 1-9.
Sack M., Hofbauer A., Fischer R. & Stoger, E. (2015). The increasing value of plant-made proteins. Current Opinion in Biotechnology 32: 163-170.
Sarker A., Rathore AS. & Gupta, RD. (2019). Evaluation of scFv protein recovery from E. coli by in vitro refolding and mild solubilization process. Microbial Cell Factories 18(5).
Schmidt GW. & Delaney, SK. (2010). Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Molecular Genetics and Genomics 283(3): 233-241.
Schouten A., Roosien J., Bakker J. & Schots, A. (2002). Formation of disulphide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol. The Journal of Biological Chemistry 277(31): 19339-19345.
Shoji Y., Jones RM., Mett V., Chichester JA., Musiychuk K., Sun X., Tumpey TM., Green BJ., Shamloul M., Norikane J., Bi H., Hartman CE., Bottone C., Stewart M., Streatfield SJ. & Yusibov, V. (2013). A plant-produced H1N1 trimeric
hemagglutinin protects mice from a lethal influenza virus challenge. Human, Vaccines, and Immunotherapeutics 9(3): 553-560.
Stelzer S., Basso W., Silvan JB., Ortega-Mora LM., Maksimov P., Gethman J., Conraths FJ. & Schares, G. (2019). Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact. Food and Waterborne Parasitology 15(e00037).
Takeno K. (2012). Stress-induced flowering. In H Hirt and K Shinozaki (Eds.), Abiotic Stress Responses in Plants (pp.331-345). New York, NY: Springer.
Takeno K. (2016). Stress-induced flowering: the third category of flowering response. Journal of Experimental Botany 67(17): 4925-4934.
Takeyama N., Kiyono H. & Yuki, Y. (2015). Plant-based vaccines for animals and humans: recent advances in technology and clinical trials. Therapeutic Advances in Vaccine 3(5-6): 139-154.
Tamura M., Togami J., Ishiguro K., Nakamura N., Katsumoto Y., Suzuki K., Kusumi T. & Tanaka, Y. (2003). Regeneration of Verbena (Verbena x hybrida) by Agrobacterium tumefaciens. Plant Cell Reports 21(5): 459-466.
Tizaoui K., & Kchouk, ME. (2012). Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco. Genetics and molecular biology 35(3): 640–649.
Wang JL., Zhang NZ., Li TT., He JJ., Elsheikha HM. & Zhu, XQ. (2019). Advances in the development of anti-Toxoplasma gondii vaccines: Challenges, opportunities, and perspectives. Trends Parasitology 35(3): 239-253.
Wang XF., Li L., Yang T., Lui J., Fan Y., Zhu X. & Wang, XZ. (2015). Single-chain variable fragment (scFv) expression in tobacco plants via agroinoculation. Russian Journal of Plant Physiology 62(3): 401–407.
Wang ZD., Liu HH., Ma ZX., Ma HY., Li ZY., Yang ZB., Zhu XQ., Xu B., Wei F. & Liu, Q. (2017). Toxoplasma gondii Infection in Immunocompromised Patients: A Systematic Review and Meta-Analysis. Frontiers in Microbiology 8(389).
Wen-Bin Y., Jin-Lei W., Qian G., Yang Z., Kai C., Qing L., Qin-Li L., Xing-Quan Z. & Dong-Hui Z. (2019). Immunization With a Live-Attenuated RH:ΔNPT1 Strain of Toxoplasma gondii Induces Strong Protective Immunity Against Toxoplasmosis in Mice. Frontiers in Microbiology 10: 1875-1887.
Yao J., Weng Y., Dickey A. & Wang, KY. (2015). Plants as factories for Human Pharmaceuticals: Applications and Challenges. International Journal of Molecular Science 16(12): 28549-28565.
Yuan X., Chen X., Yang M., Hu J., Yang W., Chen T., Wang Q., Zhang X., Lin R. & Zhao, A. (2016). Efficient construct of a large and functional scFv yeast display library derived
from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated
recombination. Applied of Microbiology and Biotechnology 100(9): 4051-4061.
Yusakul G., Sakamoto S., Juengwatanatrakul T., Putalun W., Tanaka H. & Morimoto, S. (2015). Preparation and application of a monoclonal antibody against the isoflavone glycoside daidzin using a mannich reaction‐derived hapten conjugate. Phytochemical Analysis 27(1): 81-88.
Zhou X., Carranco R., Vitha S. & Hall, TC. (2005). The dark side of green fluorescent protein. New Phytologist 168: 313-322.
Zhou X., Chandrasekharan MB. & Hall, TC. (2004). High rooting frequency and functional analysis of GUS and GFP expression in transgenic Medicago truncatula A17. New Phytologist 162: 813-822.