模拟真菌毒素表位的嵌合核酸适配子的构建及无毒、环保真菌毒素检测体系的建立

Mycotoxins are secondary metabolites mainly produced by different fungal species when they contaminate in different agro-products. They occur frequently in various food and feed commodities globally, leading to animal and human health risks and causing global concern on the food safety issues. More than four hundred mycotoxins have been identified and reported and the key important mycotoxins which are highly prevalent in the contaminated agro-food products are aflatoxins (AFs), deoxynivalenol (DON), fumonisins (FBs), zearalenone(ZEN), etc. Most mycotoxins are immunosuppressive agents and some are carcinogens, hepatotoxins, nephrotoxins, neurotoxins and reproductive toxicity. Accurate monitoring is the best way to control and reduce mycotoxin exposure. However, in the process of immunoassay monitoring, mycotoxins and conjugated toxin are still widely used as standard analytes or competitive antigen, which could harm the operators and contaminate the environment as the secondary pollution. .So, this project employ the SELEX (systematic evolution of ligands by exponential enrichment ) technology to screen the aptamers which can mimic the epitopes of aflatoxin B1/M1,zearalenone, fumonisin B1, and deoxynivalenol, etc., respectively. The targets are the F(ab)2 fragments of the specific monoclonal antibody against the mycotoxin. The aptamers characteristics such as the specificity, affinity, Kd, stability, conformational were analyzed using the surface plasmon resonance technique. The core functional domain will be determined using site-directed mutation approach. And then the site specified chemical modification will be carried out to improve the stabilities of the aptamers. Chimeric aptamer will be constructed employing many different possible types of chimerization strategies to generate more stable and efficient aptamers with aptamer–aptamer, aptamer–biomacromolecules and aptamer–nanoparticle. These reconstructed chimeras can mimic several different mycotoxins epitopes. The enzyme-linked oligonucleotide assays (ELONA) were developed using the single or chimeric aptamers which could replace the mycotoxins and conjugated toxins. Moreover, based on the ELONA, series of analytical methods, including immunochromatographic assay, immunomagnetic assay, and suspension array will be prepared, which have the advantages of rapidity, high sensitivity, and high-throughput, respectively. These methods can meet the needs for different purpose and different samples. A hypotoxic, environmental friendly testing platform will be built for the analysis of mycotoxins and other low weight molecules which having toxicity and harmful for human and animal. This project will establish a new immunological detection direction and provide strong technical supports for food safty.

真菌毒素具有细胞毒性、生殖毒性和免疫毒性，由其导致的农畜产品安全问题引发全球关注。目前在真菌毒素免疫学检测中普遍使用真菌毒素标准品或偶联毒素，给操作人员和环境造成严重的二次污染。为解决该问题，本项目以真菌毒素单克隆抗体为靶标，采用SELEX技术，分别筛选模拟4种真菌毒素表位的特异性单链DNA核酸适配子；应用SPR技术评估适配子的特异性、亲和力、解离常数，并解析其构象特征；通过核苷酸定点突变探索适配子的核心功能域，根据功能域寡核苷酸组成进行相应的化学修饰，获得更稳定、特异的适配子；将适配子与适配子、适配子与生物分子进行嵌合，构建模拟多种真菌毒素的嵌合适配子，进而替代相应的真菌毒素，建立基于免疫学原理的真菌毒素检测体系：实时快速的寡核苷酸免疫层析法、痕量的免疫磁酶分析法、高通量的寡核苷酸液相芯片等，创建真菌毒素的无毒、环保检测平台，为保障农畜产品安全和环境友好提供新的技术支持，项目具有原创性。

真菌毒素具有细胞毒性、生殖毒性和免疫毒性，由其导致的农畜产品安全问题引发全球关注。目前在真菌毒素免疫学检测中普遍使用真菌毒素标准品或偶联毒素，给操作人员和环境造成严重的二次污染。为解决该问题，本项目以真菌毒素玉米赤霉烯酮单克隆抗体为靶标，采用SELEX技术，筛选到模拟玉米赤霉烯酮表位的特异性单链DNA核酸适配子，并对适配子的特异性、亲和力、解离常数，并解析其构象特征。体外人工合成一个全长71个碱基、中间随机序列为35nt的ssDNA文库，采用SELEX技术，以玉米赤霉烯酮单克隆抗体为靶分子，进行15轮筛选，并利用生物素-链霉亲和素-辣根过氧化物酶系统测定每一轮富集文库与ZEN单抗的亲和力，将富集文库进行克隆、测序，并运用DNAMAN软件分析适配子序列的一级结构和二级结构，测定克隆子与玉米赤霉烯酮单克隆抗体的亲和力，并采用筛选适配子替代毒素建立相应的检测方法。经过SELEX 15轮的筛选，筛96个适配子进行克隆和序列分析，对序列正确的适配子进行二级结构分析和亲和性检测。获得序列全长71bp的63个适配子，软件分析预测其二级结构主要为茎环状结构，说明茎环结构是适配子与靶分子结合的基础。其中46号适配子亲和力较高，以该适配子建立酶联寡核苷酸试验(ELOSA)检测方法。方阵滴定确定的最佳条件为：ZEN单抗最佳包被浓度为1μg/孔，适配子1:10稀释后浓度为80ng/ml，辣根过氧化物酶标的链霉亲和素1:300稀释后浓度为3μg/ml。批内差异7.6%，批间差异9.3%，差异率均小于10%，表明该方法有较好的可靠性，玉米赤霉烯酮单克隆抗体特异性适配子的获得为今后研制低毒、环保的免疫学检测试剂盒提供了前提条件。同时基于实验室的单克隆抗体，建立了针对FB1、DON和ZEN三种真菌毒素的一站式快速侧向层析免疫分析法，检出限分别为 60、12.5、 6 ng/mL。建立了基于量子点荧光纳米微球的定量免疫荧光检测法，检测范围分别为2.295 ng/mL- 69.867 ng/mL (FB1)、0.465 ng/mL-16.19 ng/mL (DON)、 0.295 ng/mL-2.575 ng/mL (ZEN)，与HPLC-MS/MS的检测结果吻合性好。搭建了真菌毒素无毒、环保的多种检测平台，为保障农畜产品安全和环境友好提供新的技术支持。