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Transfer domain of HIV-1 Tat mediates HSV-1 thymidine kinase into hepatic cancer cells: the mediating effect and efficacy
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Abstract:
Objective： To explore the killing effect of the fusion protein (HSV1-TK fused with an 11-amino-acid peptide of the basic domain of the HIV-1 Tat protein, Tat11) on hepatoma cell line HepG2. Methods：Two single oligonucleotide chains encoding HIV-Tat47-57（Tat11）were synthesized and were used for producing double strand oligonucleotide through introducing BamHⅠ and HindⅢ and annealing. 15% non-denaturing polyacrylamide gel electrophoresis was used for analysis of the annealing result. The full length cDNA encoding HSV1-TK was amplified from r-pAs16Dr by PCR. Then the 2 fragments were sub-cloned into a preukaryotic expression vector (pET-32c). A single colony of E. coli BL21 containing the plasmid pET-32c-Tat11-TK was innoculated LB broth, diluted 1/100 into 1 000 ml LB broth, and was then treated with 1 mmol/L IPTG. The recombinant Tat11-TK was purified with Ni2+ chelating HiTrap HP column and its intercellular translocation ability was evaluated by immnunohistochemistry and Western blot. Results： The recombinant Tat11-TK protein showed bands at about 60 700, which was in accordance with the theoretical value of the fusion protein, also proved the presence of inclusion body. The result of immnunohistochemistry showed that Tat11-TK could bind to the cell surface and Western blot showed that it could also effectively enter into the HepG2. It was also found that HepG2 was more sensitive to GCV（150 μmol/L） in the presence of Tat11-TK. Conclusion： The fused protein Tat11-TK can be highly expressed in a preukaryotic system and has potent ability for membrane perforation and enzyme trafficking.
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