新型肽适体对豇豆花叶病毒的高效纯化。

Efficient Purification of Cowpea Chlorotic Mottle Virus by a Novel Peptide Aptamer.

机构信息

Federal Institute for Materials Research and Testing (BAM), 12489 Berlin, Germany.

Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 10117 Berlin, Germany.

出版信息

Viruses. 2023 Mar 7;15(3):697. doi: 10.3390/v15030697.

Abstract

The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications.

摘要

豇豆花叶病毒(CCMV)是一种被探索用于纳米技术平台的植物病毒。其外壳蛋白的强大自组装机制允许药物封装和靶向递送。此外,外壳纳米颗粒可用作可编程平台来展示不同的分子部分。鉴于未来的应用,植物病毒的高效生产和纯化是关键步骤。在既定的方案中,由于成本、难以扩展和安全问题,需要超速离心是一个重大限制。此外,最终病毒分离物的纯度往往不清楚。在此,开发了一种从感染植物组织中纯化 CCMV 的先进方案,重点是效率、经济性和最终纯度。该方案涉及使用 PEG 8000 沉淀,然后使用新型肽适体进行亲和提取。使用尺寸排阻色谱法、MALDI-TOF 质谱法、反相 HPLC 和夹心免疫测定法验证了该方案的效率。此外,通过 HPLC 和在 220nm 处检测,证明亲和柱的最终洗脱液具有极高的纯度(98.4%)。我们提出的方法的扩大似乎很直接,这为大规模生产此类纳米材料开辟了道路。这种高度改进的方案可能会促进植物病毒作为体外和体内应用的纳米技术平台的使用和实施。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ecf0/10051510/47b298a002aa/viruses-15-00697-g001.jpg

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