Construction of NK4 gene lentiviral vector and its expression in bone mesenchymal stem cells / 生物医学工程学杂志

ABSTRACT

The present investigation was to construct lentiviral vector carrying the human gene NK4 and transfect the human bone mesenchymal stem cells (hBMSCs) and to determine the expression of NK4 gene in hBMSCs after transfection. The NK4 gene was obtained from HGF cDNA by polymerase chain reaction(PCR), and the pGC-FU-NK4 plasmid was constructed by double restriction enzyme digestion and gene recombinant. The titer of virus was tested by real-time quantitative PCR. After transfected by lentivirus, the green fluorescent protein (GFP) in hBMSCs was observed using fluorescence microscope, and the expression of NK4 in culture supernatant was detected by enzymelinked immunosorbent assay (ELISA). The sequence of the PCR product was consistent with the data of GeneBank by DNA sequencing. The virus titer was 2 X 10(8)TU/ml. Strong green fluorescence was observed in the cell membrane and cytoplasm of hBMSCs with fluorescent microscopy. The expression of NK4 in culture supernatant was increased with time extension. The hBMSCs can be transfected by NK4 gene expressing lentiviral vector safely and effectively, and the expressin and secretion of NK4 was persistent and stable.