Construction of staphylococcal enterotoxin A eukaryotic expression plasmid and its expression in laryngocarcinoma Hep-2 cell-derived exosomes

Abstract

Objective To construct pmiR-RB-REPORT-SEA-GPI, the eukaryotic expression plasmid of staphylococcal enterotoxin A (SEA) and glycosylphosphatidylinositol (GPI) , transfect human laryngocarcinoma Hep-2 cells with this plasmid, and determine the expression of SEA in exosomes secreted by Hep-2 cells. Methods The cohesive end of prokaryotic plasmid pGSI-SEA-GPI was digested with NOT I. Separation and purification of the target gene SEA-GPI was completed by agarose gel electrophoresis. The eukaryotic expression plasmid pmiR-RB-REPORT-SEA-GPI was constructed by ligating the eukaryotic expression plasmid pmiR-RB-REPORT with SEA-GPI using T4 ligase. Hep-2 cells were transfected with pmiR-RB-REPORT-SEA-GPI by using lipofectamine method. The expression of SEA-GPI mRNA in Hep-2 cells was detected by fluorescent quantitative real-time PCR (qRT-PCR) . The exosomes secreted by Hep-2 cells were extracted and determined for expression of SEA by Western blotting. Results Agarose gel electrophoresis showed that the eukaryotic expression plasmid pmiR-RB-REPORT-SEA-GPI was successfully constructed. qRT-PCR showed that SEA-GPI mRNA was expressed in Hep-2 cells transfected with pmiR-RB-REPORT-SEA-GPI. Western blotting showed SEA protein expression in exosomes secreted by Hep-2 cells. Conclusion SEA may be anchored to exosomes membrane via GPI structure, which provides a basis for preparation of SEA-eoxsome tumor vaccines containing SEA-GPI anchoring protein. Key words: Staphylococcal enterotoxin A; Glycosylphosphatidylinositols; Transfection; Exosomes; Fluorescent quantitative real-time PCR