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A New Strategy to Identify Naturally Presenting SLA-I Bound Peptides Derived from the O Serotype of Foot-and-Mouth Disease Virus, by Mild Acid Elution in a VP1 Stably Expressed PK15 Cell Line

Year: 2025

Type: article

Abstract: Multi-epitopes of FMDV can be used to develop a novel vaccine. Determining how to screen naturally presenting epitope peptides derived from FMDV is crucial for advancing progress in this area. In this study, a transient expression plasmid named pEGFP-N1-VP1 was transfected into Porcine Kidney Epithelial cells 15 (PK15). The positive cells that stably expressed the O-VP1 gene of FMDV were screened with gradient concentrations of G418 (Geneticin). The constructed pEGFP-N1-VP1/PK15 cell line was eluted by pH 3.3 phosphate buffer to isolate the eluted peptides, followed by desalting, liquid chromatography–tandem mass spectrometry (LC–MS/MS), a flow cytometric analysis of SLA-I expression, and an ELISA detection of SLA-I bound peptides. It was demonstrated that a PK15 cell line stably expressing the VP1 gene was initially screened out at 500 μg/mL of G418, followed by culturing at 300 μg/mL. The O-VP1 expression was identified using an image analysis system, RT-PCR, and Western blot analysis. Thirty-seven peptides derived from O-VP1 were eluted from the constructed cell line. The flow cytometric analysis and ELISA detection results showed that the eluted peptides were associated with SLA-I and bound. This is the first known study to construct a cell line for screening naturally presenting antigenic peptides derived from the O serotype of FMDV.

Source: Animals

DOI: 10.3390/ani15213097