Cellfectin reagent

Sf9 cells, a clonal isolate of Spodoptera frugiperda Sf21 cells (IPLB-SF21-AE), were grown in T25 cell culture flasks with complete growth medium (Sf-900 II SFM, Invitrogen, Carlsbad, CA, USA) at 27°C without CO₂, and the cells were diluted 1:3 when they covered the bottom of the flask. The cells in the logarithmic growth phase were transfected with the recombinant baculovirus bacmid DNA encoding anti-Aβ scFv using the Cellfectin reagent (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. The supernatant containing recombinant budded viruses, designated P1, were harvested 72 h after infection and centrifuged at 500 × g for 5 min to remove cellular debris. Generally, the P1 viruses were amplified through three consecutive rounds of Sf9 cell infection at a high multiplicity of infection (MOI, 20 plaque-forming units per cell) to obtain the P3 virus.

cDNA sequences encoding complete presumptive AaChE1 constructs were cloned into pBlueBac4.5/V5-His baculoviral expression plasmid (Invitrogen, Carlsbad, CA, USA) using InFusion cloning (In-Fusion^® HD Cloning Kit, Takara Bio, San Jose, CA, USA) essentially as previously described [39 (link)]. PCR was initiated by 30 s at 98 °C followed by 35 cycles of 10 s at 98 °C, 10 sec at 60 °C, and 2 min at 72 °C. The expression constructs were cotransfected with Bac-N-Blue DNA (Invitrogen, Carlsbad, CA, USA) and Cellfectin reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions into 5 × 10⁶ Sf21 insect cells seeded onto 100 mm plates and overlaid with 1.5% BacPlaque agarose (Novagen, Millipore Sigma, Burlington, MA, USA) in Grace’s medium containing 150 µg/mL of Bluo-gal (5-bromo-3-indolyl β-D-galactopyranoside). Blue plaques were picked and used to infect 25–50% confluent Sf21 cells in 5 mL of Sf900 III SFM insect medium (Thermo Fisher Scientific, Waltham, MA USA). AChE activity of culture supernatant was monitored daily beginning 2 d after picking plaques and continuing until ChE activity reached a maximum or complete cell death in the plaque culture flasks. Baculoviral-expressed recombinant AaChE1 preparations were harvested as baculoviral culture supernatants.

The full-length gene encoding the receptor-binding domain of hemagglutinin (HA1) from influenza virus strain A/wild-duck/Korea/ES/2004 (H5N2) was obtained, as previously described [14] (link). The HA1 gene was amplified by PCR and digested with XhoI and HindIII and then subcloned into the pBAC6 baculovirus transfer vector (Novagen, Darmstadt, Germany), which contained 6 His-tag at the N-terminal and signal peptides for protein secretion in insect cells.
Recombinant pBAC6 plasmids and linearized baculovirus DNA were co-transfected into Sf21 insect cells, as described in the BD BaculoGold baculovirus expression system protocols (BD Biosciences, Franklin Lakes, NJ). Briefly, recombinant pBAC6/HA plasmids (2 µg) and linearized baculovirus DNA (0.5 µg) were mixed in Cellfectin reagent (Invitrogen, Carlsbad, CA) for 5 min and then added to 1 ml of Sf-900 serum-free media. After 15 min of incubation at room temperature, the DNA mixture was added to the Sf21 cells (2.0×10⁶ cells/ ml) in the T25 flask and incubated at 28°C for 4 h while rocking back and forth. Following the rocking incubation, the DNA mixture was removed, and 4 ml of fresh Sf-900 serum-free media was added to insect cells. The insect cells were then incubated at 28°C for 4 days, and the supernatant, which was enriched with recombinant baculovirus (pBAC6/HA), was collected by centrifugation at 1000 × g for 5 min.