DataSheet1_Production of Potyvirus-Derived Nanoparticles Decorated with a Nanobody in Biofactory Plants.PDF

Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses—the largest group of RNA viruses that infect plants—decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini (Cucurbita pepo) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes.

病毒纳米颗粒（Viral nanoparticles, VNPs）近年来受到广泛关注，可作为构建新型材料的基础单元，满足纳米技术与医药领域中一系列潜在功能需求。病毒衣壳是展示小型表位的理想载体，可将表位插入所选外壳蛋白（coat protein, CP）的合适位点。表面展示抗体的VNPs被认为是治疗与诊断领域极具应用前景的工具。由于体积小巧，纳米抗体是经典抗体用于表面展示的理想替代方案。纳米抗体源自骆驼科（Camelidae）动物的重链抗体可变域（VHH），具备体积小、结构简单、亲和力与特异性高等多项适宜用作治疗分子的特性。
本研究通过基因编码手段，制备了两种源自马铃薯Y病毒属（该类病毒是感染植物的最大RNA病毒类群）的VNPs，并以纳米抗体对其进行修饰。我们在西葫芦（Cucurbita pepo）植株中构建了经抗绿色荧光蛋白（green fluorescent protein, GFP）纳米抗体修饰的南瓜黄色矮化花叶病毒（zucchini yellow mosaic virus, ZYMV）来源VNPs。如其他病毒的相关研究所示，此类经纳米抗体修饰的ZYMV来源VNPs的表达，需在融合蛋白间插入小RNA病毒（picornavirus）2A剪接肽，最终得到未修饰外壳蛋白与修饰外壳蛋白混合的种群。
我们同时在本氏烟（Nicotiana benthamiana）植株中制备了经同一抗GFP纳米抗体修饰的烟草蚀刻病毒（tobacco etch virus, TEV）来源VNPs。值得注意的是，本案例中无需借助2A剪接肽，直接将纳米抗体与病毒外壳蛋白融合即可完成VNPs组装，理论上可得到完全修饰的VNPs。
针对两种表达体系，我们均通过透射电子显微镜（transmission electron microscope）证实了重组VNPs的正确组装与纯化；通过蛋白质免疫印迹（western blot）与结合实验验证了融合有纳米抗体的外壳蛋白的功能活性。
综上，本研究报道了经纳米抗体修饰的植物来源基因编码VNPs的制备方法。该体系可作为在植物中可持续生产含纳米抗体的纳米材料的理想替代方案，用于诊断与治疗领域。