The DNA construct (nef-vpr-gp160-p24) was digested from the pUC57 cloning vector usingBamHI andHindIII restriction enzymes (Thermo Fisher Scientific) and subcloned into theBamHI andHindIII cloning sites of pcDNA3.1(−) eukaryotic expression vector (Invitrogen) using T4 DNA ligase (Thermo Fisher Scientific). The pcDNA3.1 (−) vector harboring thenef-vpr-gp160-p24 gene construct was transformed into the competent DH5αE. coli using heat shock, and ampicillin-resistant colonies were selected. The pcDNA-nef-vpr-gp160-p24 was extracted using the Plasmid DNA Extraction Mini Kit (FAVORGEN, Taiwan) and purified by Xtra Maxi Plus Endotoxin-Free kit (MACHEREY-NAGEL, Germany). The concentration and purity of pcDNA-nef-vpr-gp160-p24 were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The presence of thenef-vpr-gp160-p24 gene was confirmed by digestion with the restriction enzymes and sequencing. The pcDNA3.1 (−) vector harboring the HIV-1nef gene was previously prepared by our group (Kadkhodayan et al.2017).

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