Isopropyl β d 1 thiogalactopyranoside (iptg)

The nef-vif-gp160-p24 and nef-vpu-gp160-p24 genes from pUC57 vectors were inserted into BamHI and HindIII restriction sites of the pET24a (+) vector, a T7 promoter based plasmid (Invitrogen, USA) as shown in Fig 4B. The E. coli strains of BL21 (DE3) and Rosetta (DE3) were used as expression hosts. These hosts were transformed with the expression plasmids harboring the genes of interest. The transformants were selected on Luria-Bertani (LB) agar plate and inoculated into 3 ml of Luria-Bertani (LB) broth containing 100 μg/ ml kanamycin (Sigma). The culture was shaken at 37°C overnight, and then transferred to Ty2x medium. Once the cell density (OD) at wavelength of 600 nm reached 0.7–0.8, the expression was induced by adding 1 mM IPTG (SinaClon bioscience Co, Iran), and the induced culture was shaken for 1, 2, 3, 4 and 16 h under the same conditions. The cell pellets were collected and analyzed by SDS-PAGE in a gel containing 12% (W/V) polyacrylamide (SDS gel apparatus; BioRad), followed by staining with coomassie brilliant blue. Also, the expression was confirmed by western blotting using anti-His antibody (Abcam, USA).

The material in this study included GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia), Clone JET PCR Cloning Kit (Thermo Fisher Scientific, Inc., USA), pET28a, expression vector (Novagen, USA), IPTG (Sinaclon, Iran), kanamycin (Sigma, Germany), Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA), Ni–NTA agarose (Qiagen, USA), Anti-His antibody (Bioligand, USA), IgG-horseradish peroxidase (HPR)-conjugated secondary antibody (Abcam, USA), EDC (G-Biosciences, USA), SANH (Bioworld, USA), FITC (Sigma-Aldrich, USA), 3-MA (Sigma, Germany), and rapamycin (Sigma, Germany).

The pET22b (+)-anti-EpEX-scFv expression plasmid developed in our previous works was transformed into expression hosts [7 ]. A single colony of E. coli harboring pET22b (+)-anti-EpEX-scFv was inoculated into 3 mL of ampicillin (100 μg/mL)-supplemented LB medium. After overnight constant shaking at 37 °C, the culture was transferred to LB medium supplemented with ampicillin at a ratio of 1∶10. To induce the expression of anti-EpEX-scFv, 0.8 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) (Sinaclon, Iran) was added to the culture in cell density between 0.7 and 0.9. The mixture was shaken at 24 h at 37 °C.