Pbudce4.1 plasmid

The HEK-293 T cells were transfected with the pBudCE4.1 plasmid (Invitrogen) containing the DNA fragment encoding HA0 of the A/Washington/05/2011(H1N1) strain. The cells were cultured in 6-well plates and transfected with the plasmids using the Lipofectamine transfection reagent (Invitrogen). At 24 h after transfection, the cells were treated with TPCK-treated trypsin (2.5 μg ml⁻¹) for 10 min at 37 °C to cleave HA0 into HA1 and HA2. Afterwards, the cells were washed with PBS to decrease the pH to 5.0. Syncytia formation was apparent. To investigate the neutralizing mechanism of 3E1, the cells were incubated with 100 μg ml⁻¹ 3E1 or 7F2 (neutralizing antibody against HCV) for 30 min at 37 °C after the trypsin digestion, respectively, and then fixed with 4% (w/v) paraformaldehyde and stained with 1% (w/v) toluidine blue in PBS. The cells were observed with an optical microscope and imaged. The experiment was replicated three times at least.

SEGFP contains a signal peptide from the human IgG kappa chain in the N-terminal. The expression cassette was composed of a CMV promoter, SEGFP coding region, and BGH polyA sequence.
To construct a TNFR-Fc expression control plasmid, a DNA fragment of the human EF1α promoter and TNFR-Fc gene was amplified by PCR from the proprietary TNFR-Fc expression vector. The amplified DNA fragment was digested with PvuI/EcoRI, and then inserted into a pBudCE4.1 plasmid (Invitrogen, USA) to replace the CMV promoter.
All HR plasmids were constructed from the control vector. For HR plasmid A, the full-length CMV sequence and 400 bp SEGFP N-terminal region were amplified, digested by PvuI, and then inserted upstream of the human EF1α promoter. The 300 bp SEGFP C-terminal sequence and BGH polyA signal sequence were cloned and inserted downstream of the zeocin-resistance gene after digestion by NheI.
HR plasmid B was obtained by ligating the NheI-digested SEGFP c-polyA-Neo p fragment into digested plasmid A. The SEGFP c-polyA-Neo p fragment contained the SEGFP C-terminal sequence region (300 bp) to the partial Neo gene.
HR plasmid C was also derived from plasmid A, in which the upstream HR element was replaced by a partial CMV promoter (CMV p; 200 bp).