Nanobody technology: feeding target authentication and mitigation strategies in crop protection.

未来作物保护战略发展的一个重要目标是选择害虫物种而不是非害虫物种。基于蛋白质结构的生物制剂提供了一条潜在的途径。开发破坏分子的抗体技术是一种很有吸引力的方法。传统抗体是具有复杂生物发生机制的低聚蛋白。然而，相对较大的抗体尺寸限制了组织进入和穿透目标部位。在本提案中，我们将研究纳米体：抗体可变重链的单链简单多肽当量(1)。它们具有表位特异性，可以选择性地靶向分子并执行功能扰动。纳米体简化了重组表达，并提供了易于通过转基因方法引入生物系统的干扰技术。我们提出了利用秀丽隐杆线虫的实验可追溯性来翻译纳米体技术的实验。得天独厚的秀丽隐杆线虫。秀丽隐杆线虫是一种细菌捕食者，无论是自由重组纳米体还是表达纳米体的细菌，都可以通过取食的方式启动试剂递送（2,3）。我们将使用已建立的表型，具有众所周知的分子决定因素，其破坏在定量行为分析中得分。这些表型反映了已建立的化学缓解害虫的破坏。此外，秀丽隐杆线虫允许对系统进行遗传操作，用人工标签表达候选目标分子。针对这些标签的商业纳米体可以询问新目标或途径的潜力。化学生物学支持纳米体研究。完全体外方法可以识别候选纳米体并编码cdna。商业纳米体到工程目标中的人造标签将用于启动工作。已知的拓扑结构和控制功能或蛋白-蛋白相互作用的关键结构域将优化功能干扰纳米体候选表位的筛选(5)。研究和调节纳米体的分布。一个重要的目标是验证纳米体作为生物制剂可以用于作物保护。蠕虫提供了一个机会来探索在复杂生物体的背景下如何接近目标。我们将测试纳米体工程表达表位识别有或没有细胞和组织穿透标签(6)。这些短片段的工程序列已经发现，通过喂食天然或修饰的实体，纳米体或修饰的纳米体进入生物体的渗透程度比较高(7)。模型秀丽隐杆线虫的发展将突出试剂和方案，以便在更多遗传上易于处理的害虫物种中进行进一步测试。卓越的价值，在允许细胞不渗透实体获得通道跨越细胞边界。

An important goal for the future development of crop protection strategies is selective targeting of pest species over non-pest species. Biologics based on protein architectures offer a potential route. The development of antibody technologies that disrupt molecules is an attractive approach. Conventional antibodies are oligomeric proteins with complex biogenesis. However, the relatively large size of antibodies restricts tissue access and penetration to target sites. In this proposal, we will investigate nanobodies: single chain simple polypeptide equivalents of the antibody variable heavy chain (1). These have epitope specificity to selectively target molecules and execute functional perturbation. The nanobodies have simplified recombinant expression and provide interfering technologies readily introduced into biological systems by transgenic approaches. We propose experiments designed to translate nanobody technology by taking advantage of the experimental tractability of C. elegans.1. Advantaged by C.elegans. C. elegans are bacterivores advantaging feeding based approaches whether free recombinant nanobodies or bacteria expressing nanobodies initiate reagent delivery (2,3). We will use established phenotypes that have well known molecular determinants whose disruption is scored in quantitative behavioural assays. These phenotypes mirror disruptions played out in established chemical mitigation of pests.Furthermore, C. elegans allows the genetic manipulation of the system to express candidate target molecules with artificial tags. Commercial nanobodies against such tags can interrogate the potential of novel targets or pathways.2. Chemical Biology to support nanobody investigation. Fully in-vitro approaches can identify candidate nanobodies and encoding cDNAs. Commercial nanobodies to artificial tags in engineered targets will be used to initiate work. The known topology and critical domains that control function or protein-protein interaction will optimize the screening of candidate epitopes for function perturbing nanobodies (5).3. Investigating and modulating nanobody distribution. An important goal is validation of the nanobodies as biologics that can be used in crop protection.The worm provides an opportunity to probe how accessible targets are in context of a complex organism. We will test nanobodies engineered to express epitope recognition with and without a cell and tissue penetrating tags (6). These short stretches of engineered sequence have found compare penetration of nanobodies or modified nanobodies into the organism by feeding with native or modified entity (7). Development in the model C. elegans will highlight reagents and protocols for further testing in more genetically tractable pest species. excellent value in allowing cell impermeable entities to gain passage across cellular boundaries.