黄曲霉毒素生物合成关键调控因子AFLR的免疫上转换荧光胞内实时动态示踪方法研究

Among all the mycotoxins that contaminate agricultural products, alfatoxins are found to be the most toxic so far. AFLR is the key regulatory factor for aflatoxin biosynthesis. Due to the lack of real-time dynamic tracking method for AFLR at the subcellular level, It becomes the bottleneck to study the relationship between the subcellular localization of AFLR and its regulatory function on aflatoxin-producing ability of Aspergillus flavus. For this reason, the applicant have developed two nanobodies for AFLR and synthesized upconverting nanoparticles exhibiting superior fluorescence characteristics, such as good photochemical stability and capable of overcoming background fluorescence interference in biological matrix. This project is proposed to further synthesize and screen UCNPs with good biocompability and fluorescence properties, and then assemble them with the developed nanobodies to obtain highly specific immuno-upconversion fluorescence probes for AFLR. To develop real-time dynamic tracking method for AFLR at the subcellular level, the structure-activity relationship of the immuno-upconversion fluorescence probes is studied to explore the optimal application conditions. Finally, in order to verify the scientificness and practicability of the developed real-time dynamic tracking method and calibrate its application conditions, the influence rule of the spatial distribution of intracellular AFLR towards the aflatoxin-producing ability of Aspergillus flavus is tried to be studied and revealed. The developed real-time dynamic tracking method for intracellular AFLR will provide a scientific approach for the in-depth analysis of AFLR regulatory function on aflatoxin biosynthesis, which is of great significance to study on the early warning, prevention and control of aflatoxin contamination in agricultural products.

黄曲霉毒素是迄今发现污染农产品毒性最强的一类真菌毒素，AFLR是黄曲霉毒素生物合成途径中的关键调控因子，因缺乏AFLR亚细胞水平实时动态示踪方法，成为研究AFLR亚细胞定位与黄曲霉菌产毒力之间关系的瓶颈。为此，申请人前期研制出AFLR特异性纳米抗体2种，合成出具有良好光化学稳定性和抗生物基质背景荧光干扰特性的上转换荧光纳米材料（UCNPs），本项目拟在此基础上，进一步合成筛选兼具良好生物相容性和荧光性能的UCNPs，并与自主AFLR纳米抗体组装成特异性上转换荧光免疫探针；通过研究探针构效关系探明最佳应用条件，建立AFLR亚细胞水平实时动态示踪方法；最后尝试利用该方法研究揭示AFLR在黄曲霉菌细胞内空间分布对菌产毒力的影响规律，校准方法应用条件，验证所建立方法的科学性和实用性，从而为深入剖析AFLR调控黄曲霉毒素生物合成机制提供科学方法支撑，对农产品黄曲霉毒素污染预警与防控研究具有重要意义。

黄曲霉毒素是迄今发现污染农产品毒性最强的一类真菌毒素，严重威胁人类健康与生命安全，制约农业产业化发展和国际贸易。AFLR是黄曲霉毒素生物合成中的关键调控因子，因缺乏AFLR细胞水平示踪方法成为研究AFLR与黄曲霉菌产毒力之间关系的瓶颈。本项目提出了开展黄曲霉毒素生物合成关键调控因子荧光免疫探针胞内示踪方法研究，主要内容包括：设计和研制了具有良好生物相容性和荧光性能的上转换荧光纳米材料4种；研究探明了荧光纳米材料与黄曲霉毒素生物合成关键调控因子纳米抗体高效组装方式，在此基础上研制出兼具特异性和高灵敏的荧光免疫探针5种；通过探针构效关系研究建立了黄曲霉毒素生物合成关键调控因子黄曲霉菌细胞内示踪方法；并选取不同产毒力的黄曲霉菌株，进一步阐明了黄曲霉毒素生物合成关键调控因子与菌株产毒力的相关性，为深入剖析AFLR调控黄曲霉毒素生物合成机制提供了科学方法支撑，对农产品黄曲霉毒素污染预警与防控研究具有重要意义。受该项目资助，已发表论文6篇，其中SCI论文5篇；申请发明专利6项，获授权发明专利2项；参编著作1部。

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