严重急性呼吸综合征冠状病毒2（SARS-CoV-2）慢病毒假型的生产、滴定、中和、储存及冻干
Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes.
作者信息
Di Genova Cecilia, Sampson Alex, Scott Simon, Cantoni Diego, Mayora-Neto Martin, Bentley Emma, Mattiuzzo Giada, Wright Edward, Derveni Mariliza, Auld Bethany, Ferrara Bill T, Harrison Dale, Said Mohamed, Selim Arwa, Thompson Elinor, Thompson Craig, Carnell George, Temperton Nigel
机构信息
Viral Pseudotype Unit (VPU Kent), Medway School of Pharmacy, University of Kent and Greenwich at Medway, Chatham Maritime, Kent, UK.
Laboratory of Viral Zoonotics, Department of Veterinary Medicine, Cambridge University, Madingley Road, Cambridge, UK.
出版信息
Bio Protoc. 2021 Nov 5;11(21):e4236. doi: 10.21769/BioProtoc.4236.
Abstract
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.
摘要
本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生产和滴定携带严重急性呼吸综合征冠状病毒2（SARS-CoV-2）刺突蛋白（以及D614G或其他关注变体，VOC）的高滴度病毒假型颗粒，并用于在表达血管紧张素转换酶2（ACE2）和跨膜丝氨酸蛋白酶2（TMPRSS2）的靶细胞中进行中和试验。它还提供了关于通过基因克隆替换新的刺突变体的详细说明，以及关于冻干和储存/运输的考虑因素，以便广泛应用。使用本方案获得的结果表明，SARS-CoV-2假型颗粒的生产滴度与SARS-CoV和中东呼吸综合征冠状病毒（MERS-CoV）假型颗粒相当，可被人康复血浆和单克隆抗体中和，并可在一系列实验室温度下储存和冻干，以便分发和后续应用。
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