力达霉素-胰岛素样生长因子1融合蛋白的构建及其抗非小细胞肺癌活性研究

发布时间：2018-03-05 20:05

本文选题：胰岛素样生长因子　切入点：力达霉素　出处：《中国药学杂志》2016年12期 　论文类型：期刊论文

【摘要】：目的制备一种由胰岛素样生长因子1(insulin-like growth factor 1,IGF-1)和力达霉素(lidamycin,LDM)构成的融合蛋白,并对其抗非小细胞肺癌(non-small cell lung cancer,NSCLC)活性进行检测。方法合成融合蛋白基因ldp-igf并将其插入到p ET30a载体中,再将获得的重组表达载体p ET30-ldp-igf转化至大肠杆菌中进行诱导表达,得到融合蛋白LDP-IGF。采用细胞免疫荧光实验和基于流式细胞术的亲和实验检测LDP-IGF蛋白与非小细胞肺癌细胞的结合能力;力达霉素的发色团(AE)与LDP-IGF蛋白在体外进行分子组装,获得强化融合蛋白LDP-IGF-AE。四甲基偶氮唑蓝法实验检测LDP-IGF-AE对不同非小细胞肺癌细胞的体外杀伤活性,PI染色结合流式细胞术检测LDP-IGF-AE对细胞周期的影响以及Annexin V-FITC/PI双染法检测LDP-IGF-AE对非小细胞肺癌细胞凋亡的诱导作用。结果经过诱导LDP-IGF蛋白在大肠杆菌中以包涵体形式表达。采用Ni~(2+)亲和层析技术对包涵体蛋白进行分离纯化,经过变性和分步透析复性后获得了高纯度、有活性的融合蛋白LDP-IGF。亲和实验结果显示,LDP-IGF蛋白与非小细胞肺癌细胞具有高度的亲和力。与AE分子进行组装后获得的强化融合蛋白LDPIGF-AE对不同的非小细胞肺癌细胞系均有非常强烈的杀伤活性,且其杀伤活性显著高于力达霉素。细胞周期阻滞和细胞凋亡实验结果显示,极低浓度的LDP-IGF-AE处理可使非小细胞肺癌细胞阻滞于G_2/M期,并可显著地诱导细胞发生凋亡。结论融合蛋白LDP-IGF-AE对非小细胞肺癌细胞有强大的体外抗肿瘤活性,具有发展成为非小细胞肺癌靶向治疗剂的潜能。
[Abstract]:Objective to prepare a fusion protein composed of insulin-like growth factor (IGF-1) and lidamycinia (LDM-1), and to detect its activity in non-small cell lung cancer (NSCLC). Methods the fusion protein gene ldp-igf was synthesized and inserted into p ET30a vector. The recombinant expression vector p ET30-ldp-igf was transformed into Escherichia coli for induction expression. The fusion protein LDP-IGF was obtained. The binding ability of LDP-IGF protein to non-small cell lung cancer cells was detected by cell immunofluorescence assay and affinity assay based on flow cytometry. The chromophore of lidamycin (Lidamycin) and LDP-IGF protein were assembled in vitro. The enhanced fusion protein LDP-IGF-AE.Tetramethylazolium blue assay was used to detect the cytotoxicity of LDP-IGF-AE to different non-small cell lung cancer cells in vitro. The effect of LDP-IGF-AE on cell cycle was detected by flow cytometry and Annexin V-FITC / Pi double staining method was used to detect the cell cycle. Results the LDP-IGF protein was expressed as inclusion body in E. coli. Ni~(2 affinity chromatography was used to isolate and purify the inclusion body protein. After denaturation and stepwise dialysis renaturation, high purity was obtained. The results of affinity assay showed that LDP-IGF protein had a high affinity with non-small cell lung cancer cells. The enhanced fusion protein LDPIGF-AE obtained by assembly with AE molecules on different non-small cell lung cancer cell lines. Have very strong killing activity, The results of cell cycle arrest and apoptosis test showed that very low concentration of LDP-IGF-AE could block NSCLC cells in G _ 2 / M phase. Conclusion the fusion protein LDP-IGF-AE has strong antitumor activity against non-small cell lung cancer cells in vitro and has the potential to become a target therapeutic agent for non-small cell lung cancer.
【作者单位】：新乡医学院;河南省分子诊断与医学检验技术协同创新中心;新乡医学院第一附属医院;中国医学科学院北京协和医学院医药生物技术研究所;
【基金】：国家自然科学基金资助项目(81202447)
【分类号】：R96

【相似文献】