Human parvovirus B19 virus-like particle formation in Nicotiana benthamiana

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS

Protein expression and purification Pub Date : 2024-10-31 DOI:10.1016/j.pep.2024.106616

Sakika Kimura , Jiahui Ong , Atsushi Kasai , Shinji Akada , Hirotaka Ebina , Michiko Sasabe , Eiji Morita

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Abstract

There has been a surge in the interest to utilize plants as hosts for producing vaccine antigens. In this study, we demonstrated the successful expression of the human parvovirus B19 (B19V) capsid protein (VP2) in Nicotiana benthamiana cells. The B19V VP1 and VP2 genes were cloned under the control of estrogen-inducible promoters and transiently expressed in N. benthamiana leaves using the agroinfiltration method. The addition of estrogen significantly boosted the expression of VP2. Furthermore, codon optimization of the VP2 sequence resulted in over a 30-fold increase in its expression compared with that of the wild-type. Analysis of negatively stained samples by sucrose density gradient ultracentrifugation and electron microscopy revealed that the expressed VP2 proteins formed spherical particles with diameters of approximately 20 nm. Immunostaining analysis of protoplasts derived from VP2-expressing N. benthamiana leaves indicated that VP2 signals were predominantly localized in the cytoplasm. These findings strongly suggested that B19V VP2 assembles and formed virus-like particles (VLPs) within the cytoplasm of N. benthamiana cells, presenting a promising method for producing B19V VLPs in plant systems.

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人副病毒 B19 病毒样颗粒在烟草中的形成。

利用植物作为宿主生产疫苗抗原的兴趣日益高涨。在这项研究中，我们证明了人副病毒 B19（B19V）荚膜蛋白（VP2）在烟草根细胞中的成功表达。在雌激素诱导启动子的控制下克隆了 B19V VP1 和 VP2 基因，并采用农渗法在烟草叶片中进行了瞬时表达。加入雌激素可明显促进 VP2 的表达。此外，对 VP2 序列进行密码子优化后，其表达量比野生型提高了 30 多倍。通过蔗糖密度梯度超速离心和电子显微镜对负染样品进行分析，发现表达的 VP2 蛋白形成了直径约为 20 纳米的球形颗粒。对来自表达 VP2 的 N. benthamiana 叶片的原生质体进行的免疫染色分析表明，VP2 信号主要定位于细胞质中。这些发现有力地表明，B19V VP2 在 N. benthamiana 细胞的细胞质中组装并形成病毒样颗粒（VLPs），为在植物系统中生产 B19V VLPs 提供了一种可行的方法。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.