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| Screening and Activity Identification of Humanized Single Domain Antibodies Against Cadherin-like Protein of Cnaphalocrocis medinalis |
| JIANG Li-Peng1, LI Yi-Hang1, HU Jia-Meng1, HU Xiao-Dan2, LIU Bei-Bei2, LIU Xian-Jin1,2,* |
1 College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;
2 Institute of Food Satefy and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China |
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Abstract Bacillus thuringiensis (Bt) Cry toxin gene has been used to cultivate insect-resistant crop varieties, which has brought great demonstration effects to the application of protein insecticidal materials. Cadherin is one of the potential receptors of Cry toxin, carrying out target screening is of great significance for the screening and creation of novel insecticidal protein materials. In this study, Humanized Single Domain Antibody Library was screened to generate single-domain antibodies against cadherin-like protein of rice leaf roller (Cnaphalocrocis medinalis) with cadherin-like protein toxin binding region (TBR) as the antigen by phage display technique. The substructive selection strategy was used to enrich phage single domain antibodies that specifically recognized TBR protein after 4 rounds of adsorption-elution-amplification. Single colonies were randomly picked from the 4th round of screening and identified by monoclonal ELISA. Four positive clones containing the complete single domain antibody gene fragment were obtained. After sequencing and amino acid sequence alignment, 3 positive clones with different sequences were finally obtained (B4, C11 and E8). The 3 positive clones identified by Western blot and ELISA binding tests had high binding activity to cadherin-like protein from rice leaf roller. Competitive inhibition test showed that 100 μg/mL Cry1Ac could inhibit 28.6% binding of C11 to the cadherin-like protein of rice leaf roller. In this study, phage display technique was used to screen phage single domain antibodies against cadherin-like protein of rice leaf roller, which could provide material basis for further development of mimics with Cry toxin functional and biological activities.
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Received: 09 October 2019
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Corresponding Authors:
*jaasliu@jaas.ac.cn
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[1] 崔华清, 王清明. 2005. 基于重链抗体构建的单域抗体研究进展[J]. 生物工程学报, 21(3): 497-501.
(Cui H Q, Wang Q M.2005. Progress in single-domain antibody derived from heavy-chain antibody[J]. Chinese Journal of Biotechnology, 21(3): 497-501.)
[2] 康晓圳, 曹佳莉, 张保惠, 等. 2018. 单域抗体的研究和应用进展[J]. 生物工程学报, 34(12): 1974-1984.
(Kang X Z, Cao J L, Zhang B H, et al.2018. Single-domain antibody-advances in research and application[J]. Chinese Journal of Biotechnology, 34(12): 1974-1984.)
[3] 徐重新, 张霄, 刘媛, 等. 2012. 抗苏云金芽孢杆菌Cry1B毒蛋白质单链抗体的筛选与鉴定[J]. 江苏农业学报, 28(4):886-890.
(Xu C X, Zhang X, Liu Y, et al.2012. Screening and identification of single-chain antibodies (scFvs) against Bacillus thuringiensis Cry1B toxin[J]. Jiangsu Journal of Agricultural Sciences, 28(4): 886-890.)
[4] 杨扬, 张大鹏, 吴兰, 等. 2009. 抗CD20人源化抗体的制备及生物活性鉴定[J]. 现代免疫学, 29(6): 491-496.
(Yang Y, Zhang D P, Wu L, et al.2009. Preparation and characterization of a humanized anti-human CD20 monoclonal antibody[J]. Current Immunology, 29(6): 491-496.)
[5] Arenas I, Bravo A, Soberon M, et al.2010. Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin[J]. The Journal of Biological Chemistry, 285(17): 12497-12503.
[6] Conrath K E, Lauwereys M, Wyns L, et al.2001. Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs[J]. Journal of Biological Chemistry, 276(10): 7346-7350.
[7] Dorsch J A, Candas M, Griko N B, et al.2002. Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca sexta: Involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis[J]. Insect Biochemistry and Molecular Biology, 32(9): 1025-1036.
[8] Dumoulin M, Last A M, Desmyter A, et al.2003. A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme[J]. Nature, 424(14): 783-788.
[9] Fabrick J A, Mathew L G, Tabashnik B E, et al.2011. Insertion of an intact CR1 retrotransposon in a cadherin gene linked with Bt resistance in the pink bollworm, Pectinophora gossypiella[J]. Insect Molecular Biology, 20(5): 651-665
[10] Fabrick J A, Tabashnik B E.2007. Binding of Bacillus thuringiensis toxin Cry1Ac to multiple sites of cadherin in pink bollworm[J]. Insect Biochemistry and Molecular Biology, 37(2): 97-106.
[11] Gomez I, Arenas I, Benitez I, et al.2006. Specific epitopes of domains Ⅱ and Ⅲ of Bacillus thuringiensis Cry1Ab toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta[J]. The Journal of Biological Chemistry, 281(45): 34032-34039.
[12] Khajuria C, Buschman L L, Chen M S, et al.2011. Identification of a novel aminopeptidase P-like gene (OnAPP) possibly involved in Bt toxicity and resistance in a major corn pest (Ostrinia nubilalis)[J]. PLOS ONE, 6(8): e23983.
[13] Muyldermans S.2001. Single domain camel antibodies: Current status[J]. Molecular Biotechnology, 74(4): 277-302.
[14] Pacheco S, Gomez I, Arenas I, et al.2009. Domain Ⅱ loop3 of Bacillus thuringiensis Cry1Ab toxin is involved in a 'ping pong' binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors[J]. The Journal of Biological Chemistry, 284(47): 32750-32757.
[15] Pardo-Lopez L, Soberon M, Bravo A.2013. Bacillus thuringiensis insecticidal three-domain Cry toxins: Mode of action, insect resistance and consequences for crop protection[J]. FEMS Microbiology Reviews, 37(1): 3-22.
[16] Rajamohan F, Lee M K, Dean D H.1998. Bacillus thuringiensis insecticidal proteins: Molecular mode of action[J]. Progress in Nucleic Acid Research and Molecular Biology, 60: 1-27.
[17] Turner K B, Liu J L, Zabetakis D, et al.2015. Improving the biophysical properties of anti-ricin single-domain antibodies[J]. Biotechnology Reports, 6: 27-35.
[18] Vachon V, Laprade R, Schwartz J L.2012. Current models of the mode of action of Bacillus thuringiensis insecticidal crystal proteins: A critical review[J]. Journal of Invertebrate Pathology, 111(1): 1-12.
[19] Wittrup K D.1999. Phage on display[J]. Trends in Biotechnology, 17(11): 423-424.
[20] Zhang J, Li Q, Nguyen T D, et al.2004. A pentavalent single-domain antibody approach to tumor antigen discovery and the development of novel proteomics reagents[J]. Journal of Molecular Biology, 341(1): 161-169.
[21] Zhang J, Valianou M, Simmons H, et al.2013. Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens[J]. FASEB Journal, 27(2): 581-589.
[22] Zhang X, Candas M, Griko N B, et al.2006. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis[J]. Proceedings of the National Academy of Sciences of the USA, 103(26): 9897-9902. |
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