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
AbstractBacillus 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.
JIANG Li-Peng,LI Yi-Hang,HU Jia-Meng, et al. Screening and Activity Identification of Humanized Single Domain Antibodies Against Cadherin-like Protein of Cnaphalocrocis medinalis[J]. 农业生物技术学报, 2020, 28(5): 884-891.
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