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Cry2Aa毒素是一种新型生物农药,分析该毒素与特异性单链抗体分子互作,并建立快速检测毒素残留的方法对保障食品和生态安全有重要意义。本研究以同源蛋白为模板对单链抗体进行建模,并进行了Cry2Aa毒素与单链抗体的分子对接模拟,确定关键结合位点,以此为基础将单链抗体作为检测抗体,建立了间接竞争时间分辨荧光免疫分析方法,对大米样品中Cry2Aa毒素进行了检测。利用生物信息学工具模拟获得单链抗体三维结构模型,分子对接结果显示重链可变区81NY82和121SGNY124区域及轻链可变区的245YSSN248氨基酸残基在与毒素结构Ⅱ区识别过程中起关键作用,为建立高效检测方法奠定基础,进一步基于该单链抗体建立的时间分辨检测方法灵敏度(IC10)为0.08 ng/m L,中抑制浓度(IC50)为7.99 ng/m L,线性检测范围(IC20~IC80)为0.24~263.77 ng/m L,且与常规双抗夹心ELISA法检测呈良好线性关系。
Cry2Aa toxin is a novel biopesticide. It is important to analyze the interaction of this toxin with specific single chain antibody molecules and to establish a rapid method to detect toxin residues in food and ecological security. In this study, single-chain antibodies were modeled using a homologous protein as a template, and the molecular docking simulation of Cry2Aa toxins was performed to determine the key binding sites. Based on this, single-chain antibodies were used as detection antibodies to establish Indirect competitive time-resolved fluorescence immunoassay was used to detect Cry2Aa toxin in rice samples. The three-dimensional structure model of single chain antibody was obtained by bioinformatics tools. The results of molecular docking showed that the amino acid residues 245YSSN248 of the heavy chain variable region 81NY82 and 121SGNY124 and the light chain variable region play a key role in the recognition of the toxin Ⅱ region (IC10) was 0.08 ng / mL and the IC50 was 7.99 ng / mL. The linearity of detection range (IC20 ~ IC80) ranged from 0.24 to 263.77 ng / m L, and showed a good linear relationship with routine double-antibody sandwich ELISA.