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. Author manuscript; available in PMC: 2019 Nov 25.
Published in final edited form as: Vaccine. 2019 Jun 11;37(30):4184–4194. doi: 10.1016/j.vaccine.2019.04.071

Fig. 1.

Fig. 1.

Construction and expression of polycistronic plasmids expressing four KSHV glycoproteins. (a) Schematic representation of pCAGGS-gpK8.1-F-2A-gB-F-2A-WTgL-2A-gH-F−/+HR2 plasmids (not drawn to scale). A single transcript was synthesized to express the gpK8.1 ectodomain (ED) fused to NDV fusion protein (F) transmembrane/cytoplasmic (TM/CT) domains (gpK8.1-F), the gB ED fused to NDV F-TM/CT (gB-F), and the gH ED fused to NDV F-TM/CT (gH-F), each with or without heptad region 2 (+/−HR2), and with full-length wild-type gL (WTgL) inserted between the gB and gH sequences. Sequence fidelity was confirmed using Sanger sequencing. The numbers shown represent corresponding amino acid numbers of the EDs. (b) Expression of gpK8.1-gB-WTgL-gH−/+HR2 in HEK-293 and CHO cells. 106 cells from HEK-293 or CHO cells were seeded in a six-well plate and transfected with 2 µg pCAGGS-gpK8.1-F-2A-gB-F-2A-WTgL-2A-gH-F−/+HR2 or pCAGGS-gpK8.1-F+HR2 (positive control). Transfection efficiency was evaluated 48 h post-transfection by staining transfected HEK-293 and CHO cells with gpK8.1 mAb, followed by secondary antibody goat anti-mouse IgG conjugated to AF488. Unstained cells, cells stained with secondary antibody alone (isotype control), and un-transfected cells served as negative controls, and gpK8.1-transfected cells served as positive control. Cells were analyzed using FACS by acquiring 10,000 events.