采用弱阴离子交换整体柱对肠道病毒 71 进行浓缩和纯化。
Concentration and purification of enterovirus 71 using a weak anion-exchange monolithic column.
机构信息
Animal Health Biotechnology, Temasek Lifesciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
出版信息
Virol J. 2014 May 27;11:99. doi: 10.1186/1743-422X-11-99.
BACKGROUND
Enterovirus 71 (EV-71) is a neurotropic virus causing Hand, Foot and Mouth Disease (HFMD) in infants and children under the age of five. It is a major concern for public health issues across Asia-Pacific region. The most effective way to control the disease caused by EV-71 is by vaccination thus a novel vaccine is urgently needed. Inactivated EV-71 induces a strong, virus-neutralizing antibody response in animal models, protecting them against a lethal EV-71 challenge and it has been shown to elicit cross-neutralizing antibodies in human trials. Hence, the large-scale production of purified EV-71 is required for vaccine development, diagnosis and clinical trials.
METHODS
CIM® Monolith columns are single-piece columns made up of poly(glycidyl methacrylate co-ethylene dimethacrylate) as support matrix. They are designed as porous channels rather than beads with different chemistries for different requirements. As monolithic columns have a high binding capacity, flow rate and resolution, a CIM® DEAE-8f tube monolithic column was selected for purification in this study. The EV-71 infected Rhabdomyosarcoma (RD) cell supernatant was concentrated using 8% PEG 8000 in the presence of 400 mM sodium chloride. The concentrated virus was purified by weak anion exchange column using 50 mM HEPES + 1 M sodium chloride as elution buffer.
RESULTS
Highly pure viral particles were obtained at a concentration of 350 mM sodium chloride as confirmed by SDS-PAGE and electron microscopy. Presence of viral proteins VP1, VP2 and VP3 was validated by western blotting. The overall process achieved a recovery of 55%.
CONCLUSIONS
EV-71 viral particles of up to 95% purity can be recovered by a single step ion-exchange chromatography using CIM-DEAE monolithic columns and 1 M sodium chloride as elution buffer. Moreover, this method is scalable to purify several litres of virus-containing supernatant, using industrial monolithic columns with a capacity of up to 8 L such as CIM® cGMP tube monolithic columns.
背景
肠道病毒 71 型（EV-71）是一种嗜神经病毒，可导致 5 岁以下婴幼儿患手足口病（HFMD）。它是亚太地区公共卫生关注的主要问题。控制 EV-71 引起的疾病的最有效方法是接种疫苗，因此急需一种新的疫苗。在动物模型中，灭活的 EV-71 可诱导强烈的、中和病毒的抗体反应，使它们免受致命的 EV-71 攻击，并且在人体试验中已显示出可诱导交叉中和抗体。因此，需要大规模生产纯化的 EV-71 用于疫苗开发、诊断和临床试验。
方法
CIM® Monolith 柱由聚（甲基丙烯酸缩水甘油酯-co- 二乙烯基苯）作为支撑基质组成的单件式柱。它们被设计为具有不同化学性质的多孔通道而不是珠子，以满足不同的要求。由于整体柱具有高结合能力、流速和分辨率，因此在本研究中选择 CIM® DEAE-8f 管整体柱进行纯化。在 400 mM 氯化钠存在下，使用 8% PEG 8000 浓缩 EV-71 感染的横纹肌肉瘤（RD）细胞上清液。使用 50 mM HEPES + 1 M 氯化钠作为洗脱缓冲液，通过弱阴离子交换柱对浓缩病毒进行纯化。
结果
通过 SDS-PAGE 和电子显微镜证实，在 350 mM 氯化钠浓度下获得了高度纯净的病毒颗粒。通过 Western blot 验证了病毒蛋白 VP1、VP2 和 VP3 的存在。整个过程的回收率达到 55%。
结论
使用 CIM-DEAE 整体柱和 1 M 氯化钠作为洗脱缓冲液，通过一步离子交换色谱法可回收高达 95%纯度的 EV-71 病毒颗粒。此外，该方法可扩展至使用工业用整体柱（如 CIM® cGMP 管整体柱），以 1 M 氯化钠作为洗脱缓冲液，从几升含病毒的上清液中纯化病毒。
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