title: 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.
authors.affiliation: Departments of Molecular Biology, College of Basic Medical Sciences, Jilin University, Changchun, China.
authors.forename: Mingli
authors.initials: M
authors.lastname: Fang
authors.forename: Wenzhen
authors.initials: W
authors.lastname: Diao
authors.forename: Boqi
authors.initials: B
authors.lastname: Dong
authors.forename: Hongfei
authors.initials: H
authors.lastname: Wei
authors.forename: Jialin
authors.initials: J
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Hua
authors.forename: Miaomin
authors.initials: M
authors.lastname: Zhang
authors.forename: Sheng
authors.initials: S
authors.lastname: Guo
authors.forename: Yue
authors.initials: Y
authors.lastname: Xiao
authors.forename: Yongli
authors.initials: Y
authors.lastname: Yu
authors.forename: Liying
authors.initials: L
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Wan
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D017925:Circovirus / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008623:Mercaptoethanol; D051379:Mice; D046529:Microscopy, Electron, Transmission; D011489:Protein Denaturation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014508:Urea; D022223:Vaccines, Subunit / Q000276:immunology / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000276:immunology / Q000378:metabolism; D058425:Vaccines, Virus-Like Particle / Q000276:immunology / Q000378:metabolism*; D014765:Viral Vaccines / Q000276:immunology / Q000378:metabolism*; D022701:Virosomes / Q000378:metabolism*
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; D058425:Vaccines, Virus-Like Particle; D014765:Viral Vaccines; D022701:Virosomes; D008623:Mercaptoethanol; D014508:Urea