食品中小分子危害物非竞争免疫分析的纳米肽高效筛选及其识别机制研究

The noncompetitive immunoassay based on nano-peptide was a new technology to monitor trace small molecule hazards in food. But restricted by nano-peptide screening system, low screening efficiency has become a bottleneck for the development this technology. In this project, Di-2-ethylhexyl phthalate (DEHP) was used as model analytes, and “DEHP-antibody” immunocomplex was used as screening ligand. Ribosome display (RD) technology and next generation sequencing (NGS) technology were combined to establish a new system for screening nano-peptide in vitro. Then, based on the selected nano-peptide and immunocomplex, the noncompetitive immunoassay technology and its evaluation system were established to determination of trace DEHP in food. In addition, with site-directed mutagenesis technology, the interaction between nano-peptide and immunocomplex was characterized by using fluorescence spectrum and circular dichroism technology. Comparison the sensitivity, affinity, interaction between nano-peptide and immunocomplex discrepancy between before and after mutation, and the recognition activity related key amino acid sites or regions were identified, which illustrated recognition mechanism between nano-peptide and immunocomplex. With development of this project, it will produce new breakthrough on improving screening efficiency of nano-peptide, and widen application scope of new noncompetitive immunoassay for the small molecule hazards; It will provide a scientific basis for the development of the recognition mechanism theory between nano-peptide and immunocomplex.

基于纳米肽的非竞争免疫分析模式是监控小分子危害物痕量残留的新技术。但受纳米肽筛选系统的限制，筛选效率低已成为制约该技术发展的瓶颈。据此本项目提出以邻苯二甲酸二（2-乙基己基）酯（DEHP）为模型，“DEHP-抗体”免疫复合物为筛选配基，运用核糖体展示技术与高通量测序技术相结合，构建高效筛选和鉴定纳米肽的新系统。进而基于高活性纳米肽和“DEHP-抗体”免疫复合物，构筑食品中痕量DEHP检测的非竞争免疫分析新方法及评价体系。此外，借助定点突变技术，利用荧光光谱和圆二色谱技术表征纳米肽与免疫复合物相互作用构象。比较突变前后纳米肽灵敏度、亲和力及相互作用构象差异，确证识别活性相关的关键氨基酸位点或区域，揭示纳米肽与免疫复合物间的识别机制。本项目将在有效改进纳米肽筛选效率上取得新突破，从而拓宽小分子危害物非竞争免疫分析新方法的应用范围；还将为纳米肽与免疫复合物识别机制理论的发展提供科学依据。

基于纳米肽的非竞争免疫分析是监控小分子危害物痕量残留的新技术。但受纳米肽筛选系统的限制，筛选效率低已成为制约该技术发展的瓶颈。本项目提出以食品包装材料中易残留的邻苯二甲酸二（2-乙基己基）酯（DEHP）为模型，“DEHP-单克隆抗体”免疫复合物为筛选配基，基于磁珠法原位核糖体展示技术构建纳米肽（14肽库）的体外筛选新系统。同时，以荧光定量PCR技术对筛选进程进行监测。进而将最后一轮富集文库进行高通量测序，选择出现频率高的序列与pLIP6-GN载体偶联，构建纳米肽融合表达系统，进而构建非竞争免疫分析新方法。结果显示，经过重叠延伸PCR构建的核糖体展示纳米肽文库约为410 bp。对构建文库的多样性测序结果显示，同源率分别为27%、31%、33%、35%、36%、38%、44%、46%，表明文库多样性良好。经8轮富集后，Q-PCR的Ct值明显减小，说明纳米肽文库得到了富集。经高通量测序结果显示，多条序列的富集次数大于100次，选择富集次数最多的20条序列进行融合表达。N3和N15序列显示出与免疫复合物有较高的结合活性，N3的SC50为57.8 ng/mL，线性范围为12 ng/mL-278.4 ng/mL。N15的SC50为72 ng/mL，线性范围为4.84 ng/mL-1070.47 ng/mL。对这两条序列中的1个氨基酸进行突变后，它们都失去与DEHP-单克隆抗体的结合能力。本项目在有效改进纳米肽筛选效率上取得新突破，从而拓宽小分子危害物非竞争免疫分析新方法的应用范围；还为纳米肽与免疫复合物识别机制理论的发展提供科学依据。