Balke Ina, Reseviča Gunta, Zeltins Andris
Latvian Biomedical Research and Study Centre Ratsupites 1 k-1, Riga, LV-1067, Latvia.
Methods Mol Biol. 2018;1776:19-34. doi: 10.1007/978-1-4939-7808-3_2.
Plant virus-like particles (VLPs) structurally resemble their progenitor viruses, but are noninfectious due to absence of viral nucleic acids. Since the 1980s, VLPs have been actively studied with the aim of constructing different nanomaterials, including immunologically active carriers for peptides and whole proteins and proteinaceous shells for the packaging of different ligands.The technological developments using VLPs require large amounts of purified particles. Here, we describe the laboratory process for isolation and purification of two unmodified plant VLPs, derived from two sobemoviruses, cocksfoot mottle virus (CfMV) and rice yellow mottle virus (RYMV), which is based on cultivation of recombinant Escherichia coli cells, VLP precipitation from bacterial extracts and ultracentrifugation. The suggested purification scheme allows the production of 4-45 mg of purified sobemoviral VLPs from a 1 l bacterial culture, depending on the required purity level. Additionally, we provide short protocols for VLP characterization using SDS-PAGE, agarose gel electrophoresis, ultraviolet and mass spectrometry, dynamic light scattering, and electron microscopy.
植物病毒样颗粒（VLP）在结构上与其原始病毒相似，但由于缺乏病毒核酸而无感染性。自20世纪80年代以来，人们一直在积极研究VLP，旨在构建不同的纳米材料，包括用于肽和全蛋白的免疫活性载体以及用于包装不同配体的蛋白质外壳。使用VLP的技术发展需要大量纯化颗粒。在此，我们描述了从两种南方菜豆花叶病毒，即鸭茅斑驳病毒（CfMV）和水稻黄斑驳病毒（RYMV）中分离和纯化两种未修饰植物VLP的实验室方法，该方法基于重组大肠杆菌细胞的培养、从细菌提取物中沉淀VLP以及超速离心。根据所需的纯度水平，建议的纯化方案可从1升细菌培养物中产生4 - 45毫克纯化的南方菜豆花叶病毒VLP。此外，我们还提供了使用SDS - PAGE、琼脂糖凝胶电泳、紫外和质谱、动态光散射以及电子显微镜对VLP进行表征的简短方案。
