An optimised method for the production of MERS-CoV spike expressing viral pseudotypes.
An optimised method for the production of MERS-CoV spike expressing viral pseudotypes.
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DOI：
10.1016/j.mex.2015.09.003
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发表时间：
2015
期刊：
影响因子：
1.9
通讯作者：
Temperton N
中科院分区：
文献类型：
--
作者：
Grehan K;Ferrara F;Temperton N
假型病毒颗粒的生产和使用已广泛用于许多病毒，并且在血清学和疫苗开发领域的应用是多方面的。病毒假型已被证明是研究病毒进化对血清学结果、病毒向性和免疫原性研究影响的有力工具。假型病毒是嵌合构建体，其中一种病毒的外层（表面）糖蛋白与另一种病毒的复制缺陷型病毒“核心”相结合。假型允许在低生物安全设施内进行准确、序列导向、敏感的抗体中和测定和抗病毒筛选，并为野生型病毒的使用提供安全有效的替代方案。这里概述的协议代表了一种快速可靠的方法，用于在慢病毒核心上生成具有 MERS-CoV 刺突蛋白的高滴度假型病毒颗粒，并改编自之前发布的协议。该方案针对在 100 mm 培养皿中转染进行了优化，收获了 7 ml 上清液，但它可以轻松扩展到不同的生产量。使用现成的试剂。一致的高病毒滴度。快速生成用于菌株变异研究的新型糖蛋白。该协议具有许多优点，包括：
The production and use of pseudotyped viral particles are widely established for many viruses, and applications in the fields of serology and vaccine development are manifold. Viral pseudotypes have proven to be powerful tools to study the effects of viral evolution on serological outcomes, viral tropism and immunogenicity studies. Pseudotyped viruses are chimeric constructs in which the outer (surface) glycoprotein(s) of one virus is combined with the replication-defective viral “core” of another virus. Pseudotypes allow for accurate, sequence-directed, sensitive antibody neutralisation assays and antiviral screening to be conducted within a low biosecurity facility and offer a safe and efficient alternative to wildtype virus use. The protocol outlined here represents a rapid and reliable method for the generation of high-titre pseudotype viral particles with the MERS-CoV spike protein on a lentiviral core, and is adapted from previously published protocols. This protocol is optimised for transfection in a 100 mm Petri dish with 7 ml of supernatant harvested, however it can be readily scaled to different production volumes. Use of readily available reagents. Consistent, high virus titres. Rapid generation of novel glycoproteins for research into strain variation. This protocol has a number of advantages including: