Recombinant trimeric IAV hemagglutinin proteins open reading frames were cloned into the pCD5 expression vector as described previously55 (link), in frame with a GCN4 trimerization motif (KQIEDKIEEIESKQKKIENEIARIKK), a superfolder GFP56 (link) and the Twin-Strep-tag (WSHPQFEKGGGSGGGSWSHPQFEK); (IBA, Germany). The open reading frames of the HAs of A/Hong-Kong/1/1968 H3 (AFG71887.1), A/Netherlands/816/1991 H3 (EPI_ISL_114608), A/Netherlands/109/2003 H3 (EPI_ISL_113016), A/Netherlands/761/2009 H3 (EPI_ISL_1107270), A/Singapore/INFH-16-0019/2016 H3 (3 C.2a) (QQY97257.1), were synthesized and codon optimized by GenScript.
The trimeric HAs were expressed in HEK293T (CRL-11268) and HEK 293 S GNTI(-) (ATCC CRL-3022) cells with polyethyleneimine I (PEI) in a 1:8 ratio (µg DNA:µg PEI) for the HAs as previously described16 (link). The transfection mix was replaced after 6 h by 293 SFM II suspension medium (Invitrogen, 11686029), supplemented with sodium bicarbonate (3.7 g/L), Primatone RL-UF (3.0 g/L, Kerry, NY, USA), glucose (2.0 g/L), glutaMAX (1%, Gibco), valproic acid (0.4 g/L) and DMSO (1.5%). Culture supernatants were harvested 5 days post-transfection and the HA0 was purified with sepharose strep-tactin beads (IBA Life Sciences, Germany) according to the manufacturer’s instructions.
The trimeric HAs were expressed in HEK293T (CRL-11268) and HEK 293 S GNTI(-) (ATCC CRL-3022) cells with polyethyleneimine I (PEI) in a 1:8 ratio (µg DNA:µg PEI) for the HAs as previously described16 (link). The transfection mix was replaced after 6 h by 293 SFM II suspension medium (Invitrogen, 11686029), supplemented with sodium bicarbonate (3.7 g/L), Primatone RL-UF (3.0 g/L, Kerry, NY, USA), glucose (2.0 g/L), glutaMAX (1%, Gibco), valproic acid (0.4 g/L) and DMSO (1.5%). Culture supernatants were harvested 5 days post-transfection and the HA0 was purified with sepharose strep-tactin beads (IBA Life Sciences, Germany) according to the manufacturer’s instructions.
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