Zhao Dandan, Sun Bo, Sun Shiyang, Fu Bin, Liu Chuntian, Liu Dawei, Chu Yanfei, Ma Youlei, Bai Lu, Wu Yongge, Zhou Yan, Su Weiheng, Hou Ali, Cai Linjun, Xu Fei, Kong Wei, Jiang Chunlai
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, China.
School of Life Sciences, Jilin Agricultural University, Changchun, China.
PLoS One. 2017 Jul 21;12(7):e0181182. doi: 10.1371/journal.pone.0181182. eCollection 2017.
Human enterovirus 71 (EV71) is a major causative pathogen of hand, foot and mouth disease (HFMD) and has caused outbreaks with significant mortality among young children in the Asia-Pacific region in recent years. Towards developing a vaccine for this disease, we have expressed and purified EV71 virus-like particles (VLPs), which resemble the authentic virus in appearance, capsid structure and protein sequence, from insect cells (Sf9) using a multistep chromatography process. We demonstrated intracellular localization of the VLPs in host cells by in situ immunogold detection, electron microscopy and immunofluorescence. Characteristics of these EV71 VLPs were studied using a variety of immunological and physicochemical techniques, which aimed to reveal that the purified EV71 VLPs have good morphology and structure consistent with natural EV71 empty capsids. Results of the amino acid analysis, SDS-PAGE, Western blotting and high-performance liquid chromatography confirmed the high purity of the EV71 VLPs. However the sedimentation coefficient of the VLPs showed that they were smaller than that of secreted EV71 VLPs purified by discontinuous cesium chloride density gradients, they were similar to the empty capsids of natural EV71 virions reported previously. Combined with the previous study that EV71 VLPs purified by a multistep chromatography process were able to elicit strong humoral immune responses in mice, our results further supported the conclusion that our EV71 VLPs had well-preserved molecular and structural characteristics. The EV71 VLPs produced from the baculovirus expression system and purified by a multistep chromatography process displayed key structural and immunological features, which would contribute to their efficacy as a HFMD vaccine.
人肠道病毒71型（EV71）是手足口病（HFMD）的主要致病病原体，近年来在亚太地区导致了多起疫情，造成幼儿高死亡率。为了开发针对这种疾病的疫苗，我们利用多步色谱法从昆虫细胞（Sf9）中表达并纯化了EV71病毒样颗粒（VLP），其在外观、衣壳结构和蛋白质序列上与天然病毒相似。我们通过原位免疫金检测、电子显微镜和免疫荧光证明了VLP在宿主细胞中的细胞内定位。使用多种免疫和物理化学技术研究了这些EV71 VLP的特性，目的是揭示纯化的EV71 VLP具有与天然EV71空衣壳一致的良好形态和结构。氨基酸分析、SDS-PAGE、蛋白质印迹和高效液相色谱的结果证实了EV71 VLP的高纯度。然而，VLP的沉降系数表明它们比通过不连续氯化铯密度梯度纯化的分泌型EV71 VLP小，它们与先前报道的天然EV71病毒粒子的空衣壳相似。结合先前的研究，即通过多步色谱法纯化的EV71 VLP能够在小鼠中引发强烈的体液免疫反应，我们的结果进一步支持了我们的EV71 VLP具有保存良好的分子和结构特征这一结论。由杆状病毒表达系统产生并通过多步色谱法纯化的EV71 VLP显示出关键的结构和免疫特征这将有助于其作为手足口病疫苗的效力。
