Construction and characterization of TK/FL recombinant protein
Objective To determine the in vitro behavior of simultaneously expressed TK and FL in cells. Methods A recombinant vector carrying herpes simplex virus thymidine kinase gene (TK), internal ribosome entry site (IRES) and Flt 3 ligand gene (FL) was constructed, and transfected into the MCF 7 tumor cells (MCF 7/TK FL). FL in the supernatant was detected by ELISA. MTT was adopted to determine the sensitivity of MCF 7/TK FL cells to GCV (ganciclovir). The apoptosis of cells was monitored using electro microscope and flow cytometry. Results MCF 7/TK FL cells expressed both TK and FL genes simultaneously. Dose dependent cell killing by a transduction of the HSV TK gene followed by GCV treatment was observed. Apoptosis was also obvious in the MCF 7/TK FL cells GCV system. The amount of FL secreted in culture supernatant of MCF 7/TK FL cells was 15.7ng/ml from 10 5 cells during 4 days, as determined by ELISA. The supernatant was able to stimulate proliferation of CD34 + cells from human cord blood. Conclusion MCF 7/TK FL cells may be used as a experimental vaccine for tumor therapy, they are also valuable in producing large quantity of FL.