Abstract：
Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers\' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.
摘要：
脱氧雪腐镰刀菌烯醇(DON)是一种广泛分布于各种食品中的霉菌毒素,严重威胁食品安全。为了最大限度地减少消费者对DON的饮食接触，迫切需要开发快速、灵敏的食品中DON检测方法。在这项研究中,开发了一种双功能单链可变片段（scFv）连接的碱性磷酸酶（ALP）融合蛋白，用于快速，灵敏地检测脱氧雪腐镰刀菌烯醇（DON）。化学合成scFv基因并通过同源重组克隆到含有ALP基因的表达载体pET25b中。原核表达,净化,和融合蛋白(scFv-ALP和ALP-scFv)的活性分析被很好地表征和进行。通过计算机辅助模拟研究了scFv与DON之间的相互作用，其中包括氢键，疏水相互作用，和范德华部队.选择显示更好的双功能活性的scFv-ALP用于开发谷物中DON的直接竞争性酶联免疫吸附测定（dc-ELISA）。dc-ELISA需要90分钟进行一次测试，并显示11.72ng/mL的半抑制浓度（IC50），其中IC50比基于scFv的dc-ELISA低3.08倍。所开发的方法显示出对DON的高选择性，并且从尖峰实验中获得了良好的准确性。此外，该方法分析的实际谷物样品的检测结果与高效液相色谱法的检测结果具有良好的相关性（R2=0.97165）。这些结果表明，scFv-ALP是开发一步比色免疫测定的有前途的双功能探针，为谷物中DON的快速、灵敏检测提供了新的策略。
