Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system.
Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system.
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DOI：
10.1371/journal.pone.0020005
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Chong PC
中科院分区：
文献类型：
--
作者：
Liu CC;Guo MS;Lin FH;Hsiao KN;Chang KH;Chou AH;Wang YC;Chen YC;Yang CS;Chong PC
肠道病毒 71 (EV71) 感染最常见的表现为儿童出疹，称为手足口病 (HFMD)，在急性感染期间可引起神经系统疾病。在本研究中，我们描述了 EV71 病毒的生产、纯化和表征，该病毒由在含有 5 g/L Cytodex 1 微载体的 5 升无血清生物反应器系统中生长的 Vero 细胞产生。当初次感染时使用 10−5 的 MOI 时，感染后 6 天病毒滴度 >106 TCID50/mL。当通过蔗糖梯度区带超速离心纯化收获的 EV71 病毒浓缩物时，分离并检测了两个 EV71 病毒级分。在 24-28% 蔗糖组分中检测到的 EV71 病毒颗粒具有直径 30-31 nm 的二十面体结构，病毒感染性和 RNA 含量较低。通过 SDS-PAGE 观察到三种主要病毒蛋白（VP0、VP1 和 VP3）。 SDS-PAGE分析显示，在含有35-38%蔗糖的级分中检测到的EV71病毒颗粒大小为33-35 nm，具有较高的病毒感染性和RNA含量，并且由四种病毒蛋白（VP1、VP2、VP3和VP4）组成。这两种病毒组分经过福尔马林灭活，并在小鼠免疫原性研究中诱导高病毒中和抗体反应。两种小鼠抗血清均识别 VP1 的免疫显性线性中和表位（残基 211-225）。这些结果为基于细胞的 EV71 疫苗开发提供了重要信息，特别是为病毒抗原定量工作标准的准备。
Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.
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影响因子：
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