乳腺癌特异性多肽介导的HSV-TK/GCV系统的构建及其靶向杀伤效应

Construction and targeted effects of breast cancer specific peptide-mediated HSV-TK/GCV anti-tumor system

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 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.