de Mello Renata Gois, Bernardino Thaissa Consoni, Guardalini Luis Giovani Oliveira, Astray Renato Mancini, Antoniazzi Marta Maria, Jared Simone Gonçalves Silva, Núñez Eutimio Gustavo Fernández, Jorge Soraia Attie Calil
Laboratório de Biotecnologia Viral, Instituto Butantan, São Paulo, SP, Brazil.
Laboratório de Biologia Estrutural, Instituto Butantan, São Paulo, SP, Brazil.
Front Pharmacol. 2023 Jun 12;14:1181566. doi: 10.3389/fphar.2023.1181566. eCollection 2023.
The Zika virus (ZIKV) infections are a healthcare concern mostly in the Americas, Africa, and Asia but have increased its endemicity area beyond these geographical regions. Due to the advances in infections by Zika virus, it is imperative to develop diagnostic and preventive tools against this viral agent. Virus-like particles (VLPs) appear as a suitable approach for use as antiviral vaccines. In this work, a methodology was established to produce virus-like particles containing the structural proteins, C, prM, and E of Zika virus produced in insect cells using the gene expression system derived from baculovirus. The vector pFast- CprME -ZIKV was constructed containing the gene sequences of Zika virus structural proteins and it was used to generate the recombinant bacmids (Bac- CprME -ZIKV) through transformation into DH10Bac cells. The Bac- CprME -ZIKV was transfected in Spodoptera frugiperda (Sf9) insect cells and batches of BV- CprME -ZIKV were obtained by infection assays using a multiplicity of infection of 2. The Sf9 cells were infected, and the supernatant was collected 96 h post-infection. The expression of the CprME -ZIKV protein on the cell surface could be observed by immunochemical assays. To concentrate and purify virus-like particles, the sucrose and iodixanol gradients were evaluated, and the correct CprME -ZIKV proteins' conformation was evaluated by the Western blot assay. The virus-like particles were also analyzed and characterized by transmission electron microscopy. Spherical structures like the native Zika virus from 50 to 65 nm containing the CprME -ZIKV proteins on their surface were observed in micrographs. The results obtained can be useful in the development path for a vaccine candidate against Zika virus.
寨卡病毒（ZIKV）感染主要是美洲、非洲和亚洲的医疗保健问题，但它的流行区域已超出这些地理区域。由于寨卡病毒感染方面的进展，开发针对这种病毒制剂的诊断和预防工具势在必行。病毒样颗粒（VLP）似乎是用作抗病毒疫苗的合适方法。在这项工作中，建立了一种方法，使用源自杆状病毒的基因表达系统，在昆虫细胞中生产含有寨卡病毒结构蛋白C、prM和E的病毒样颗粒。构建了包含寨卡病毒结构蛋白基因序列的载体pFast-CprME-ZIKV，并通过转化到DH10Bac细胞中用于产生重组杆粒（Bac-CprME-ZIKV）。将Bac-CprME-ZIKV转染到草地贪夜蛾（Sf9）昆虫细胞中，并通过使用感染复数为2的感染试验获得多批BV-CprME-ZIKV。感染Sf9细胞，并在感染后96小时收集上清液。通过免疫化学分析可以观察到CprME-ZIKV蛋白在细胞表面的表达。为了浓缩和纯化病毒样颗粒，评估了蔗糖和碘克沙醇梯度，并通过蛋白质印迹分析评估了正确的CprME-ZIKV蛋白构象。还通过透射电子显微镜对病毒样颗粒进行了分析和表征。在显微照片中观察到表面含有CprME-ZIKV蛋白的50至65纳米的球形结构，类似于天然寨卡病毒。所获得的结果可能有助于寨卡病毒候选疫苗的开发进程。
