Abstract
Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. Here, we describe a robust protocol for producing DENV VLPs from transiently-transformed or stable COS-1 cells and further provide an easily adaptable antigen purification method by sucrose gradient centrifugation.
Keywords: Dengue virus (DENV), Virus-like particle (VLP), COS-1 cell, VLP purification, Sucrose gradient centrifugation
Background
Flavivirus infection results in the production of virion particles as well as non-infectious virus-like particles (VLPs), which are composed of the pre-membrane (prM) and envelope (E) structural proteins but which lack a nucleocapsid (Allison et al., 1995). In cells transfected with plasmid DNA co-expressing the prM and E proteins, these proteins self-assemble and are secreted in the form of VLPs (Hunt et al., 2001; Chang et al., 2003; Galula et al., 2014). These VLPs have structural and physico-chemical features resembling the virion particles (Shen et al., 2018). DENV VLPs have been demonstrated to be an effective immunogen and have the potential as a subunit vaccine (Konishi and Fujii, 2002; Liu et al., 2014; Shen et al., 2018), and as the alternative serodiagnostic antigens replacing virus-infected suckling mice brain preparations (Holmes et al., 2005; Crill et al., 2009; Chao et al., 2019). Here, we provide a detailed protocol for the production and purification of DENV VLPs from COS-1 cells that can be easily adapted and which requires only basic laboratory equipment.
Materials and Reagents
Equipment
Procedure
Data analysis
Fractions collected after rate-zonal centrifugation in a 5-25% linear sucrose gradient were analyzed by antigen-capture ELISA. The average OD values from duplicates of each fraction were calculated and plotted as an absorbance curve to determine which fractions at peak OD values are to be pooled (fractions 4-7, Figure 3).
Recipes
Acknowledgments
This work was supported by Ministry of Science and Technology Taiwan (MOST-107-2313-B-005-038-MY3) and (MOST-104-2321-B-005-006). This protocol was adapted from previously published studies by Chang et al. (2000), Hunt et al. (2001) Chiou et al. (2008), Crill et al. (2009), Galula et al. (2014) and Shen et al. (2018).
Competing interests
The authors declare no conflict of interest.
References
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