Functional and binding characterization of a single chain Fv antibody to abscisic acid and conjugated abscisic acid |
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Authors: | Xiaojiao Chen Yao Lu Guiyu Tan Zhen Cao Wei Liu |
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Affiliation: | 1. College of Agriculture and Biotechnology, China Agricultural University, Beijing, China;2. Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, China;3. Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, HI, USA |
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Abstract: | In the present research, we studied the binding characterization of a single chain fragment variable (scFv) antibody S12B8 with abscisic acid (ABA) and conjugated ABA by competitive enzyme-linked immunosorbent assay (ELISA) and in silico molecular docking. The nucleotide sequences of variable light (VL) and variable heavy (VH) were obtained from hybridoma cells (F12B8), which secrete monoclonal antibodies recognizing ABA and conjugated ABA. And then the genes of VL and VH were constructed to a scFvS12B8 with a nucleotide sequence of a flexible linker (G4S)3 by splicing overlap extension PCR (SOE-PCR). The fragment of S12B8 was cloned into pMAL-c2x with a maltose binding protein tag and expressed in Escherichia coli with a calculated molecular mass of 68?kDa. The results of ELISA showed dose-dependent inhibition of the purified recombinant protein S12B8 by ABA, (ABA-ME) and ABA glucose ester tetra acetyl (ABAGE tetra acetyl). The detection limits (10% inhibition) of ABA, ABA-ME and ABAGE tetra acetyl were 19.71, 8.06 and 3.95?μg/ml, respectively. On the other hand, we investigated the interactions between S12B8 and ligands by homology modeling using RosettaAntibody and molecular docking by Discovery studio 2.5 (DS 2.5). The binding energy of ABA, ABA-ME and ABAGE when fused to the S12B8 antibody was ?37.1, ?69.6 and ?112.0?kcal/mol, respectively, which agreed well with our competitive ELSIA results. |
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Keywords: | Abscisic acid conjugated ABA recombinant antibody scFv ELISA molecular docking |
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