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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。
OBJECTIVE To prepare a fusion protein consisting of insulin-like growth factor 1 (IGF-1) and lidamycin (LDM) and to evaluate its anti-tumor activity against non-small cell lung cancer lung cancer, NSCLC) activity. Methods The fusion protein gene ldp-igf was synthesized and inserted into p ET30a vector. The resulting recombinant expression vector p ET30-ldp-igf was transformed into E. coli to induce expression. The fusion protein LDP-IGF was obtained. The binding ability of LDP-IGF protein to non-small cell lung cancer cells was examined by immunofluorescence assay and flow cytometry-based affinity assay. The levamycin chromophore (AE) and LDP-IGF protein Assembly, access to enhanced fusion protein LDP-IGF-AE. MTT assay was used to determine the in vitro cytotoxicity of LDP-IGF-AE to different non-small cell lung cancer cells. PI staining and flow cytometry were used to detect the effect of LDP-IGF-AE on cell cycle and Annexin V-FITC / PI double staining method to detect LDP-IGF-AE on non-small cell lung cancer cell apoptosis. As a result, the LDP-IGF protein was induced to express in E. coli as a inclusion body. The Ni2 + affinity chromatography was used to separate and purify the inclusion body proteins. The fusion protein LDP-IGF with high purity and activity was obtained after denaturation and fractional dialysis refolding. Affinity experiments showed that LDP-IGF protein has high affinity with non-small cell lung cancer cells. The enhanced fusion protein LDPIGF-AE obtained after assembly with the AE molecule has a very strong killing activity on different non-small cell lung cancer cell lines and its killing activity is significantly higher than that of the doxycycline. The results of cell cycle arrest and apoptosis showed that LDP-IGF-AE treatment at very low concentration could block non-small cell lung cancer cells in G 2 / M phase and induce cell apoptosis significantly. Conclusion The fusion protein LDP-IGF-AE has strong in vitro antitumor activity against non-small cell lung cancer cells and has the potential to become a targeted therapeutic agent for non-small cell lung cancer.