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目的:研究乳腺癌MDA-MB-231细胞特异性转导多肽PI介导的HSV-TK/GCV抗瘤系统对乳腺癌MDA-MB-231细胞的体外靶向杀伤作用。方法:以PCR从质粒pORF-HSV-TK中扩增目的基因PI-TK,克隆到原核表达载体pET-28a(+)中,构建pET-28a(+)-PI-TK载体,转化宿主菌,经IPTG诱导表达PI-TK融合蛋白,利用His-Tag对其进行纯化,SDS-PAGE和Western blotting鉴定PI-TK融合蛋白。将不同质量浓度的PI-TK融合蛋白与MDA-MB-231细胞共培养,联合更昔洛韦(ganci-clovier,GCV)作用后,倒置显微镜下观察细胞的形态变化,CCK-8法检测MDA-MB-231细胞的增殖。结果:成功构建了重组原核表达载体pET-28a(+)-PI-TK,获得纯化的PI-TK融合蛋白,SDS-PAGE及Western blotting鉴定PI-TK融合蛋白表达正确。单独PI-TK融合蛋白不影响MDA-MB-231细胞的形态和增殖,但PI-TK融合蛋白联合GCV能剂量依赖性靶向抑制MDA-MB-231细胞的增殖,200μg/ml PI-TK+10 mg/LGCV作用的抑制率达(68.9±7.57)%;PI-TK联合GCV抑制MDA-MB-231细胞的IC50值为152.64μg/ml。上述各作用对MDA-MB-435细胞均无影响(P<0.05)。结论:乳腺癌特异性转导多肽PI介导的HSV-TK/GCV抗瘤系统可靶向杀伤MDA-MB-231细胞。
OBJECTIVE: To study the targeted killing effect of HSV-TK / GCV anti-tumor system on breast cancer MDA-MB-231 cells induced by MDA-MB-231 cell-specific transducible polypeptide PI in breast cancer. Methods: The target gene PI-TK was amplified by PCR from the plasmid pORF-HSV-TK and cloned into the prokaryotic expression vector pET-28a (+) to construct the vector pET-28a (+ The PI-TK fusion protein was induced by IPTG, purified by His-Tag, and identified by SDS-PAGE and Western blotting. The different concentrations of PI-TK fusion protein were co-cultured with MDA-MB-231 cells. Combined with ganci-clovier (GCV), morphological changes of cells were observed under inverted microscope. -MB-231 cells. Results: The recombinant prokaryotic expression vector pET-28a (+) - PI-TK was successfully constructed and the purified PI-TK fusion protein was obtained. The expression of PI-TK fusion protein was confirmed by SDS-PAGE and Western blotting. PI-TK fusion protein alone did not affect the morphology and proliferation of MDA-MB-231 cells, but PI-TK fusion protein combined with GCV inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner. The inhibitory rate of 10 mg / LGCV was (68.9 ± 7.57)%. The IC50 of PI-TK combined with GCV for inhibiting MDA-MB-231 cells was 152.64 μg / ml. The above effects had no effect on MDA-MB-435 cells (P <0.05). CONCLUSION: The PI-mediated HSV-TK / GCV anti-tumor system targeting breast cancer-specific transduction peptide can specifically target MDA-MB-231 cells.