Authors: Mélissanne de Wispelaere ,Meret Ricklin ,Philippe Souque,Marie-Pascale Frenkiel,Sylvie Paulous,Obdulio Garcìa-Nicolàs,Artur Summerfield,Pierre Charneau ,Philippe Desprès
The antigenicity of recombinant JEV proteins was assessed by transducing HEK-293T cells with the TRIP/JEV.prME or TRIP/JEV.prME vectors (Fig 2A). An empty vector served as a control. At 48 h post-transduction, the intracellular form of the E protein was detected using the anti-E MAb 4G2 by IF assay. A similar staining pattern for E was observed in TRIP/JEV-transduced cells expressing prME or prME. Immunoblot assays using mouse anti-JEV antisera detected recombinant prM and E in lysates from HEK-293T cells transduced with TRIP/JEV vectors (Fig 2B). We observed an efficient release of E protein to the supernatants of HEK-293T cells transduced with either TRIP/JEV vectors (Fig 2C, top). The presence of prM was only detected in supernatants from cells transduced with the TRIP/JEV.prME vector (Fig 2C, top). Because JEV prM and E have the capacity to self-assemble into VLPs, we assessed whether VLPs were secreted from HEK-293T cells transduced with TRIP/JEV vectors. The VLPs were detected by immunoblot assay using anti-E mAb 4G2 and anti-JEV immune serum (Fig 2C, bottom). Extracellular JEV VLPs containing prM and E accumulated in the supernatant of HEK-293T cells transduced with TRIP/JEV.prME vector but not with TRIP/JEV.prME vector. Thus, transduction of cells with TRIP/JEV.prME vector leads to efficient secretion of recombinant JEV VLPs.
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