Construction and Biological Effects in vitro of Double-Directed Tissue-Specific HSV-tk/GCV Anti-Tumor System

摘要

Objective: To construct a specifically targeted gene delivery and expression system, and to investigate the special killing effect of the HSV-tk/GCV system on human liver cancer cells in vitro. Methods: The anti-transferrin receptor (TfR) ScFv-GAL4 fusion protein expression vector ScFv-GAL4-pET28a and the eukaryotic expression plasmid pEBAF/tk-GAL4rec were constructed by recornbinant DNA technology. After the induction by IPTG, we obtained the anti-TfR ScFv-GAL4 fusion protein as delivery vector to transfect pEBAF/tk-GAL4rec into the human liver cancer cell lines HepG2 and SMMC7721 and the human lung cancer cell line A549 that overexpress TfR via receptor-mediated endocytosis. The positive cell clones were selected by hygromycin and named HepG2/tk, SMMC7721/tk and A549/tk, respectively. Cell killing after GCV application was determined by MTT. Results: The correct structure of the ScFv-Cal4 fusion protein and the plasmid pEBAF/tk-GAL4rec was confirmed by double enzyme digestion, SDS-PAGE and sequencing. HepG2/tk cells that express alphafetoprotein (AFP) to high levels (845 ng/ml) were very sensitive to GCV, while SMMC7721/tk cells that express AFP at low levels (2 ng/ml) and AFP-negative A549/tk cells were only slightly or not sensitive to GCV. Conclusion: The double-directed and tissue-specific HSV-tk/GCV anti-tumor system shows good targeting to tumor cells.