Detection of the Assembly and Disassembly of PCV2b Virus-Like Particles Using Fluorescence Spectroscopy Analysis

Abstract

Monitoring the assembly and disassembly of virus-like particles (VLPs) is important in developing effective VLP-based vaccines. We tried to establish a simple and rapid method to evaluate the status of VLP assembly using fluorescence spectroscopic analysis (FSA) while developing a VLP-based vaccine against porcine circovirus type 2b (PCV2b). We synthesized the gene coding for PCV2b capsid protein (CP). The CP was expressed in Escherichia coli in a soluble form, dialyzed into three different buffers, and assembled into VLPs. The immunogenicity of the VLPs was evaluated by an enzyme-linked immunosorbent assay using the sera of mice immunized with inactivated PCV2b. The VLP assembly was detected using transmission electron microscopy and FSA. The assembled VLPs showed a distinct FSA curve with a peak at 320 nm. We found that the assembly status was related to the immunogenicity, fluorescence intensity, and morphology of the VLP. The FSA assay was able to monitor the various denatured statuses of PCV2b VLPs treated with β-mercaptoethanol or β-mercaptoethanol plus urea. We have demonstrated that FSA can be used to detect the assembly of PCV2b VLPs produced in E. coli. This provides a simple solution for monitoring VLP assembly during the production of VLP-based vaccines.

Construction, Characterization, and Application of a Nonpathogenic Virus-like Model for SARS-CoV-2 Nucleocapsid Protein by Phage Display

“…Currently, research on viruses and spectra, including discrimination and identification of viruses by spectra and algorithms, is commonly investigated. The techniques used in research on the spectral properties of viruses include Raman spectroscopy [ 18 , 19 , 20 ], infrared spectroscopy [ 21 , 22 , 23 , 24 , 25 ], fluorescence spectroscopy [ 26 ], laser-induced breakdown spectroscopy [ 27 ], UV/Vis spectroscopy [ 28 ], etc. Valerio et al developed an ultrafast, reagent-free, and non-destructive attenuated total reflection–Fourier transform infrared spectroscopy (ATR–FTIR) method, realizing COVID-19 virus-infected samples being stoichiometrically analyzed [ 21 ].…”

Process monitoring of virus‐like particle reassembly by diafiltration with UV/Vis spectroscopy and light scattering

“…Particle size measurements provide information on the assembly progress of the capsid proteins into VLPs. Previous publications have also reported effects of the VLP tertiary structure on ultraviolet and visible (UV/Vis) and fluorescence absorption spectra (Ausar, Foubert, Hudson, Vedvick, & Middaugh, 2006;Fang et al, 2016;Hanslip, Zaccai, Middelberg, & Falconer, 2006;Hu et al, 2011;Rajendar et al, 2013). Following a systematic approach to process monitoring, a combination of PAT sensors should be chosen which allows monitoring protein concentration, protein tertiary structure, and protein size.…”

Fluorescence signatures of SARS-CoV-2 spike S1 proteins and a human ACE-2: excitation-emission maps and fluorescence lifetimes

“…[11][12][13][14] Elastic light scattering and fluorescence have been used to observe virus particles and virus-like particles. [15][16][17] Compared to Raman spectroscopy, the LIF technique yields higher signal intensities and has been applied to detect and classify viruses with excitation in the near-ultraviolet (near-UV). 18,19 As pointed out in Ref.…”