Expression and immunogenicity of secreted forms of bovine ephemeral fever virus glycoproteins applied to subunit vaccine development
| Autor: | Garry A. Luke, Hsing-Chieh Wu, Martin D. Ryan, Yi-Ting Lo, Fiona Tulloch, Chun-Yen Chu |
|---|---|
| Přispěvatelé: | BBSRC, University of St Andrews. School of Biology, University of St Andrews. Biomedical Sciences Research Complex |
| Rok vydání: | 2021 |
| Předmět: |
Ephemeral Fever
Ephemeral Fever Virus Bovine Glycosylation G protein Protein subunit NDAS Biology Applied Microbiology and Biotechnology Epitope Epitopes Mice 03 medical and health sciences chemistry.chemical_compound SDG 3 - Good Health and Well-being Antigen Animals QR180 Immunology Glycoproteins 030304 developmental biology chemistry.chemical_classification 0303 health sciences Bovine ephemeral fever virus 030306 microbiology Immunogenicity G glycoprotein General Medicine Cell biology chemistry Cell culture Vaccines Subunit QR180 Infectious diseases Cattle Glycoprotein Vaccine Biotechnology |
| Zdroj: | Journal of Applied Microbiology. 131:1123-1135 |
| ISSN: | 1365-2672 1364-5072 |
| DOI: | 10.1111/jam.15044 |
| Popis: | This study was support by grants from the Taiwan Ministry of Science and Technology (MOST-106-2911-I-020-501; MOST-107-2313-B-020-011-MY3) and the UK Biotechnology and Biological Sciences Research Council (BB/P025080/1). Aims Vaccines for bovine ephemeral fever virus (BEFV) are available but are difficult to produce, expensive, or suffer from genetic instability. Therefore, we designed constructs encoding C-terminally truncated forms (transmembrane anchoring region deleted) of glycoproteins G and GNS such that they were secreted from the cell into the media to achieve high-level antigen expression, correct glycosylation pattern, and enable further simple purification with the V5 epitope tag. Methods and Results In this study, synthetic biology was employed to create membrane-bound and secreted forms of G and GNS glycoprotein. Mammalian cell culture was employed as an antigen expression platform, and the secreted forms of G and GNS protein were easily purified from media by using a highly effective, single-step method. The V5 epitope tag was genetically fused to the C-termini of the proteins, enabling detection of the antigen through immunoblotting and immunomicroscopy. Our data demonstrated that the C-terminally truncated form of the G glycoprotein was efficiently secreted from cells into the cell media. Moreover, the immunogenicity was confirmed in mice test. Conclusions The immuno-dot blots showed that the truncated G glycoprotein was present in the total cell extract, and was clearly secreted into the media, consistent with the western blotting data and live-cell images. Our strategy presented the expression of secreted, epitope-tagged, forms of the BEFV glycoproteins such that appropriately glycosylated forms of BEFV G protein was secreted from the BHK-21 cells. This indicates that high-level expression of secreted G glycoprotein is a feasible strategy for large-scale production of vaccines and improving vaccine efficacy. Significance and Impact of the Study The antigen expression strategy designed in this study can produce high-quality recombinant protein and reduce the amount of antigen used in the vaccine. Postprint |
| Databáze: | OpenAIRE |
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