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Construction and targeted effects of breast cancer specific peptide-mediated HSV-TK/GCV anti-tumor system
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Abstract:
Objective : To investigate the targeted killing effect of breast cancer specific peptide PI-mediated HSV-TK/GCV system against human breast cancer MDA-MB-231 cells in vitro. Methods: PI-TK gene was amplified from pORF-HSV-TK plasmid by PCR, and was re-inserted into the prokaryotic expression plasmid pET-28a (+); the pET-28a (+)-PI-TK plasmid was constructed and transfected into the host bacteria. After induction with IPTG, PI-TK fusion protein was purified by His-Tag and further identified by SDS-PAGE and Western blotting analysis. MDA-MB-231 cells were cultured with different dosages of PI-TK fusion protein; after further treatment with ganciclovier (GCV), the morphology changes of MDA-MB-231 cells were observed under inverted microscope, and the cell proliferation was evaluated by CCK-8 assay. Results: Recombinant prokaryotic expression plasmid pET-28a (+)-PI-TK was successfully constructed. The purified PI-TK fusion protein was obtained and confirmed by the SDS-PAGE and Western blotting analysis. PI-TK fusion protein alone failed to affect the morphology and proliferation of MDA-MB-231 cells, but when combined with GCV, PI-TK fusion protein specifically inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner, with inhibitory rate of 200 μg/ml PI-TK + 10 mg/L GCV being (68.9±7.57)%, and IC50 being 152.64 μg/ml, but it had no effects on MDA-MB-435 cells (P<0.05). Conclusion: The breast cancer specific peptides PI-mediated HSV-TK anti-tumor system can specifically kill MDA-MB-231 cells.
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Project Supported:
supported by the National Natural Science Foundation of China (No. 3086033), the Key Foundation of Applied Basic Research Program Yunnan Province (No. 2009CC023), and the Basic Platform Construction Program for Science and Technology in Yunnan Province (No. 2007DA006)
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