抗-Cry2Aa单链抗体分子互作及荧光免疫检测研究

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/mL，中抑制浓度(IC50)为7.99 ng/mL，线性检测范围(IC20~IC80)为0.24~263.77 ng/mL，且与常规双抗夹心ELISA法检测呈良好线性关系。

Time-resolved Fluorescence Immunoassay and Molecular Recognition Mechanism of the Single Chain Variable Fragment against the Cry2Aa Toxin

Cry2Aa toxin is a new type of biopesticide, and analysis of the interactions between this toxin and a specific single chain variable fragment (scFv) to enable the establishment of a rapid method for toxin-residue detection is important for maintenance of food and ecological security. Here, a homologous protein was used as a template for modeling the three-dimensional structure of anti-Cry2Aa scFv in order to allow simulations of molecular docking between Cry2Aa and scFv and determination of key binding sites. The results showed that svFv was suitable for use as an antibody to establish an indirect competitive time-resolved fluorescence immunoassay (IC-TRFIA), which was applied for the detection of Cry2Aa toxin in rice samples. The three-dimensional structure of scFv was modeled and refined using bioinformatics tools. The molecular-docking results showed that the amino acid residues of the heavy chain variable regions 81NY82 (VH-CDR2) and 121SGNY124 (VH-CDR3) and the light chain variable region 245YSSN248 (VL-CDR3) played important roles in the recognition of the Cry2Aa domain, providing a basis for the establishment of a highly efficient detection method. The limit of the detection was 0.08 ng/mL, the 50% inhibition rate was 7.99 ng/mL, and the linear range (IC20~IC80) was from 0.24 ng/mL to 263.77 ng/mL. Furthermore, IC-TRFIA results and those of conventional double-antibody sandwich ELISA showed good linear correlation.