抗噻虫嗪重组全长抗体的制备与特异性识别机制研究

本研究旨在制备抗噻虫嗪的重组抗体,并采用计算机辅助的同源建模和分子对接的方法解析抗体和噻虫嗪的特异性分子识别机制.首先,采用表面等离子共振技术评价了抗噻虫嗪单克隆抗体的识别性能;其次,以抗噻虫嗪杂交瘤细胞株为基因来源,经分子克隆获得了抗体可变区序列,由哺乳动物细胞HEK 293(F)体外表达成功获得了全长重组抗体;最后...

Development of anti-thiamethoxam full-length recombinant antibody and investigation of the specific recognition mechanism

In this study, the recombinant antibody against thiamethoxam was prepared and the specific molecular recognition mechanism between the antibody and thiamethoxam was studied via computer-assisted homology modeling and molecular docking. Firstly, surface plasma resonance (SPR) technique was used to evaluate the recognition features of monoclonal antibody (mAb) against thiamethoxam. Secondly, the specific variable regions of heavy and light chains (VH and VL) in anti-thiamethoxam mAb were amplified from a hybridoma cell line, and full-length recombinant antibody (rAb) was successfully in vitro expressed by mammalian cell HEK 293 (F). At last, the molecular recognition mechanism of the antibody’s high specificity and sensitivity to thiamethoxam was investigated by homology modeling and molecular docking, based on the correct sequences of VH and VL. The results showed that the mAb could specifically recognize thiamethoxam and had a high binding affinity with dissociation equilibrium constant K_D of 7.995 × 10?11 mol/L. As evaluated by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), the performance of full-length rAb was similar to that of the parental mAb, exhibiting high specificity and high sensitivity to thiamethoxam, with the IC₅₀ value of 0.41 μg/L to thiamethoxam. No cross reactivity to other neonicotinoid pesticides was observed (< 0.04%). The results demonstrated that Asn39 (L-CDR1) and the other 8 amino acid residues in the hydrophobic binding pocket interacted with the target mainly through hydrogen bond and van der Waals, which appeared to be the predominant contributor to the selectivity (specificity) of the antibody. His35 (H-CDR1) and Trp108 (H-CDR3) residues located in the VH affected on the binding affinity of the antibody against thiamethoxam. In conclusion, the full-length rAb prepared in this study can replace traditional monoclonal antibody as the core reagent to establish a variety of immunoassay methods for the detection of thiamethoxam residues in environmental samples and agricultural products. The study of the recognition mechanism can provide theoretical guidance for the further improvement of antibody affinity.