Sol Ferrero, María Victoria Batto, Matías Iván Gatto, Federico Dimase, Gustavo Helguera
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引用次数: 0
Abstract
Conventional live virus research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of coronavirus disease-19 (COVID-19), requires Biosafety Level 3 (BSL-3) facilities. SARS-CoV-2 pseudotyped viruses have emerged as valuable tools in virology, mimicking the entry process of the SARS-CoV-2 virus into human cells by expressing its spike glycoprotein in a surrogate system using recombinant plasmids. One significant application of this tool is in functional assays for the evaluation of neutralizing antibodies. Pseudotyped viruses have the advantage of being competent for only a single cycle of infection, providing better safety and versatility and allowing them to be studied in BSL-2 laboratories. Here, we describe three protocols for the detection of SARS-CoV-2 neutralizing antibodies through a pseudotyped virus assay. First, SARS-CoV-2 S pseudotyped viruses (PV SARS-CoV-2 S) are produced using a Moloney murine leukemia virus (MuLV) three-plasmid system. The plasmids are designed to express the GagPol packing proteins, enhanced green fluorescent protein (eGFP) as a readout system, and the SARS-CoV-2 S protein modified to remove the endoplasmic reticulum retention domain and to improve infection. Next, the internalization of PV SARS-CoV-2 S protein in human embryonic kidney 293T (HEK-293T) cells overexpressing angiotensin-converting enzyme 2 (HEK-293T-ACE2) is confirmed by fluorescence microscopy and quantified using flow cytometry. Finally, PV SARS-CoV-2 S is used to screen neutralizing antibodies in serum samples from convalescent COVID-19 patients; it can also be used for studying the cell entry mechanisms of different SARS-CoV-2 variants, evaluating antiviral agents, and designing vaccines. © 2024 Wiley Periodicals LLC.
Basic Protocol 1: Generation of PV SARS-CoV-2 S pseudotyped virus
Basic Protocol 2: Assay of PV SARS-CoV-2 S internalization in target cells.
Basic Protocol 3: Detection of neutralizing antibodies in serum samples.
使用 SARS-CoV-2 伪原型病毒测定法检测血清样本中的中和抗体
对严重急性呼吸系统综合症冠状病毒 2（SARS-CoV-2）（冠状病毒病-19（COVID-19）的病原体）的常规活病毒研究需要生物安全三级（BSL-3）设施。SARS-CoV-2 伪型病毒已成为病毒学的重要工具，它利用重组质粒在代用系统中表达 SARS-CoV-2 病毒的尖峰糖蛋白，从而模拟 SARS-CoV-2 病毒进入人体细胞的过程。这一工具的一个重要应用是用于评估中和抗体的功能测试。伪型病毒的优点是只需一个感染周期，安全性更高，用途更广，可在 BSL-2 实验室进行研究。在此，我们介绍通过伪型病毒检测法检测 SARS-CoV-2 中和抗体的三种方案。首先，使用莫隆尼鼠白血病病毒（MuLV）三质粒系统生产 SARS-CoV-2 S 伪型病毒（PV SARS-CoV-2 S）。质粒的设计目的是表达 GagPol 包装蛋白、作为读出系统的增强型绿色荧光蛋白（eGFP）以及经过修饰的 SARS-CoV-2 S 蛋白，以去除内质网保留结构域并提高感染率。接着，通过荧光显微镜确认了 PV SARS-CoV-2 S 蛋白在过表达血管紧张素转换酶 2（HEK-293T-ACE2）的人胚胎肾脏 293T 细胞（HEK-293T）中的内化情况，并使用流式细胞术进行了量化。最后，PV SARS-CoV-2 S 可用于筛选 COVID-19 康复期患者血清样本中的中和抗体；还可用于研究不同 SARS-CoV-2 变种的细胞进入机制、评估抗病毒药物和设计疫苗。© 2024 Wiley Periodicals LLC.基本程序 1：产生 PV SARS-CoV-2 S 伪型病毒 基本程序 2：检测 PV SARS-CoV-2 S 在靶细胞中的内化。基本方案 3：检测血清样本中的中和抗体。
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