Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes
Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes
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DOI：
10.21769/bioprotoc.4236
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发表时间：
2021-11-05
期刊：
影响因子：
0.8
通讯作者：
Temperton, Nigel
中科院分区：
文献类型：
--
作者：
Di Genova, Cecilia;Sampson, Alex;Temperton, Nigel
该方案详细介绍了一种快速可靠的方法，用于在慢病毒载体核心上用SARS-CoV-2刺突蛋白（以及D614G或其他相关变体，VOC）生产和滴定高滴度病毒伪型颗粒，并用于表达血管紧张素转换酶2 （ACE2）和跨膜丝氨酸蛋白酶2 （TMPRSS2）的靶细胞中的中和试验。此外，它还提供了通过基因克隆、冻干和广泛部署潜力的存储/运输考虑来替代新穗变体的详细说明。通过该方案获得的结果表明，SARS-CoV-2假型可以以与SARS-CoV和中东呼吸综合征冠状病毒（MERS-CoV）假型相同的滴度生产，通过人类恢复期血浆和单克隆抗体中和，并在一定的实验室温度下储存并冻干，以供分发和后续应用。
This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.