靶向性抗肿瘤融合蛋白RGD-hIL-24的构建、表达和体外活性研究

目的构建靶向性抗肿瘤融合蛋白RGD-hIL-24,并对其体外抗肿瘤效应和肿瘤靶向性进行初步研究。方法利用PCR技术将GRGDS序列融合至hIL-24的N端,并将融合基因连接至表达载体pET-22b后,在大肠杆菌BL21(DE3)内进行表达。亲和层析法纯化RGD-hIL-24,复性后采用MTT比色法、荧光染色分析其体外抗肿瘤活性,并通过细胞黏附实验评价其肿瘤靶向性。结果获得RGD-hIL-24基因,序列分析正确。SDS-PAGE和Western blot证明融合基因在大肠杆菌表达相对分子质量(Mr)约为20×103的RGD-hIL-24,占全菌蛋白的26．47％,主要以包涵体形式存在。纯化后的蛋白纯度达90％以上。复性后的RGD-hIL-24能够诱导MCF-7乳腺癌细胞凋亡,显著抑制其生长,并具有肿瘤靶向性。结论大肠杆菌成功表达RGD-hIL-24融合蛋白,体外实验证实RGD-hIL-24具有显著的抗肿瘤活性和肿瘤靶向性,为其在体内抗肿瘤效应和靶向性研究奠定了基础。

The construction and expression of tumor-targeting fusion protein RGD-hIL-24 and the study of its in vitro anti-tumor activity

Objective To construct and express tumor-targeting fusion protein RGD-hIL-24 in E. coli and to study its anti-tumor activity of inducing apoptosis and targeting tumor in vitro . Methods The fusion gene GRGDS fused with N-terminus of hTL-24 was obtained by PCR. Then it was cloned into expression vector pET-22b. RGD-hIL-24 was expressed in E. coli BL21(DE3) . The fusion protein was purified by chelating chromatog-raphy. The activity of inducing apoptosis of tumor cells was observed by MTT colorimetry and fluorescence staining. The activity of targeting tumor was analyzed by cell adhesion assay. Results The fusion gene was confirmed by gene sequencing. RGD-hIL-24 was expressed in E. coli as inclusion bodies ( Mr = 20 000) which took up to 26.47% of total somatic protein. It was identified by SDS-PAGE and Western blot. The purity of fusion protein reached over 90% . It induced the apoptosis of MCF - 7 cells and was targeted to tumor cells in vitro . Conclusion The fusion protein RGD-hIL-24 was successfully constructed and highly expressed in E. coli. It could induce tumor cells apoptosis and have the activity of tumor targeting.