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Preparation of MDA-7/IL-24-HT7 fusion protein and its apoptosis inducing activity on tumor cells
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Abstract:
Objective: To construct MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors, and prepare purified MDA-7/IL-24-HT7 fusion protein, so as to study its cellular localization and apoptosis-inducing effect on tumor cells. Methods: MDA-7/IL-24 gene was amplified by PCR and cloned into vectors containing HaloTag (HT7) to construct MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors. MDA-7/IL-24-HT7 fusion protein was induced by IPTG and further puified. Cellular localization of MDA-7/IL-24-HT7 fusion protein in tumor cells was monitored by fluorescence-marked HT7 ligands. The effects of MDA-7/IL-24-HT7 fusion protein on growth and apoptosis of tumor cells were detected by MTT and Annexin V-PI staining assays. Results: MDA-7/IL-24-HT7 prokaryotic and eukaryotic expression vectors were successfully constructed. The MDA-7/IL-24-HT7 fusion protein was mainly expressed as inclusion bodies in E.coli BL21, and localized in the endoplasmic reticulum of tumor cells. MDA-7/IL-24-HT7 fusion protein inhibited growth of tumor cells. Apoptosis rates of colon cancer HCT116 cells and hepatic carcinoma SMMC7721 cells were (34.7±1.3)% and (22.1±0.9)%, respectively, after treatment with 1mg/ml MDA-7/IL-24-HT7 for 96 h, which were significantly higher than those of untreated tumor cells (P<0.01). Conclusion: MDA-7/IL-24-HT7 fusion protein containing HaloTag (HT7) can inhibit growth and induce apoptosis of tumor cells.
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Project supported by the Foundation from State Key Laboratory of Oncogenes and Related Genes (No. 80-07-03)
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