Production of GP64-free virus-like particles from baculovirus-infected insect cells
Production of GP64-free virus-like particles from baculovirus-infected insect cells
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DOI：
10.1099/jgv.0.001002
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发表时间：
2018-02-01
影响因子：
3.8
通讯作者：
Blissard, Gary W.
中科院分区：
文献类型：
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作者：
Chaves, Lorena C. S.;Ribeiro, Bergmann M.;Blissard, Gary W.
逆转录病毒Gag蛋白经常用于产生用于各种应用的“病毒样颗粒”（VLP）。逆转录病毒Gag蛋白自组装并在质膜上出芽以形成类似于天然逆转录病毒病毒粒子但不含病毒基因组的包膜VLP。杆状病毒表达载体系统已用于表达高水平的逆转录病毒Gag蛋白以产生VLP。然而，由杆状病毒感染的昆虫细胞产生的VLP制剂通常含有相对大浓度的杆状病毒出芽病毒（BV）颗粒，其在大小和密度上与VLP相似，因此在纯化VLP时可能难以分离。此外，这些包膜VLP在VLP包膜中可能具有大量杆状病毒编码的GP 64包膜蛋白。由于VLP经常被生产用于疫苗开发，因此VLP中存在GP 64包膜蛋白以及VLP制剂中存在苜蓿银纹夜蛾多衣壳核多角体病毒BV是不希望的。在目前的研究中，我们开发了一种策略，通过在杆状病毒gp64基因缺失的情况下表达人类免疫缺陷病毒1型gag基因，减少BV和消除VLP生产中的GP64。使用GP64null重组杆状病毒，我们证明了GAG介导的VLP生产和VLP中GP64的缺失，在BV生产减少的情况下。因此，该方法代表了用于在昆虫细胞中产生VLP的显著改进的方法。
The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs. Additionally, these enveloped VLPs may have substantial quantities of the baculovirus-encoded GP64 envelope protein in the VLP envelope. Since VLPs are frequently produced for vaccine development, the presence of the GP64 envelope protein in VLPs, and the presence of Autographa californica multicapsid nucleopolyhedrovirus BVs in VLP preparations, is undesirable. In the current studies, we developed a strategy for reducing BVs and eliminating GP64 in the production of VLPs, by expressing the human immunodeficiency virus type 1 gag gene in the absence of the baculovirus gp64 gene. Using a GP64null recombinant baculovirus, we demonstrate Gag-mediated VLP production and an absence of GP64 in VLPs, in the context of reduced BV production. Thus, this approach represents a substantially improved method for producing VLPs in insect cells.