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J Appl Microbiol ; 131(3): 1123-1135, 2021 Sep.
Article in English | MEDLINE | ID: mdl-33605066

ABSTRACT

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 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.


Subject(s)
Ephemeral Fever Virus, Bovine , Ephemeral Fever , Animals , Cattle , Ephemeral Fever/genetics , Ephemeral Fever/prevention & control , Ephemeral Fever Virus, Bovine/genetics , Epitopes/genetics , Glycoproteins/genetics , Mice , Vaccines, Subunit
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