Production and Characterization of Self-Assembled Virus-like Particles Comprising Capsid Proteins from Genotypes 3 and 4 Hepatitis E Virus (HEV) and Rabbit HEV Expressed in Escherichia coli

Viruses Pub Date : 2024-08-31 DOI:10.3390/v16091400

Tominari Kobayashi, Masaharu Takahashi, Satoshi Ohta, Yu Hoshino, Kentaro Yamada, Suljid Jirintai, Putu Prathiwi Primadharsini, Shigeo Nagashima, Kazumoto Murata, Hiroaki Okamoto

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Abstract

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development.

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大肠杆菌中表达的由 3 型和 4 型戊型肝炎病毒（HEV）和兔 HEV 的囊膜蛋白组成的自组装病毒样颗粒的制备与表征

戊型肝炎病毒（HEV）基因型 3（HEV-3）和 4（HEV-4）以及兔 HEV（HEV-3ra）的人畜共患传播已有记录。针对 HEV 感染的疫苗接种依赖于荚膜（开放阅读框 2，ORF2）蛋白，该蛋白具有高度免疫原性，可激发有效的病毒中和抗体。大肠杆菌（E. coli）是生产 HEV 样颗粒（VLPs）的有效系统。然而，关于在大肠杆菌中生产这些 HEV 基因型的 ORF2 蛋白以形成 VLPs 的研究并不多。在本研究中，我们构建了 21 个重组质粒，在大肠杆菌中表达了 HEV-3、HEV-3ra 和 HEV-4 的各种 N 端和 C 端截短的 HEV ORF2 蛋白。我们成功获得了 9 个 HEV-3、2 个 HEV-3ra 和 10 个 HEV-4 ORF2 蛋白，它们主要定位于包涵体中。这些蛋白质在 4 M 尿素中溶解、过滤并进行凝胶过滤。结果显示，6 个 HEV-3、1 个 HEV-3ra 和 2 个 HEV-4 截短蛋白可以组装成 VLPs。纯化的 VLPs 分子量在 27.1 至 63.4 kDa 之间，经生物分析仪评估，纯度较高（74.7%-95.3%），每 100 mL 结核病培养基的产量为 13.9-89.6 mg。免疫电子显微镜证实这些 VLPs 来自 HEV ORF2。抗原性测试表明，这些 VLPs 具有典型的 HEV 抗原性。在 Balb/cAJcl 小鼠体内进行的免疫原性评估显示，这些 VLPs 具有很强的抗 HEV IgG 反应，突出了其作为免疫原的潜力。这些发现表明，生成的不同基因型的 HEV VLPs 可作为 HEV 研究和疫苗开发的宝贵工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Viruses

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