4.8. Construction of a Stable Cell Line Overexpressing eEF1A
To construct a stable cell line overexpressing eEF1A, the swine eEF1A gene was subcloned into the lentivirus vector pFUGW (Addgene, Cambridge, MA, USA). HEK293T cells were cotransfected with the recombinant plasmid pFUGW-eEF1A or the empty vector pFUGW, and the packaging plasmids psPAX2 (Addgene) and pMD2.G (Addgene). At 6 hpt, the medium was replaced with DMEM containing 5% FBS. After 48-h incubation, the supernatant of the cell culture was harvested and ultra-centrifuged to concentrate the recombinant lentiviruses. Subsequently, PK-15 cells grown on 6-well plates were transduced with the lentiviruses of 10 transduction units (TU) per cell. The expression of EGFP-eEF1A in the transduced PK-15 cells was examined by Western blotting using a rabbit anti-eEF1A MAb (1:2000) (catalog no. ab157455; Abcam, Cambridge, UK).