融合蛋白EGF-TCS的纯化及体外靶向杀伤肿瘤细胞的研究

Purification of EGF-TCS recombinant fusion protein and its targeting action on human tumor cells in vitro

目的在大肠杆菌中表达并纯化EGF-TCS融合蛋白,观察该融合蛋白对肿瘤细胞的靶向选择性杀伤作用。方法将重组表达质粒PQE30/EGF-TCS转化至大肠杆菌M15中,表达该融合蛋白(EGF-TCS)。产物经Ni-NTAAgrose亲和层析柱纯化;用流式细胞仪检测肿瘤细胞株和正常肝LO2细胞株的EGFR表达量;进行细胞杀伤实验,验证EGF-TCS的选择性杀伤能力;用流式细胞仪检测细胞凋亡的方法进一步考察EGF-TCS的靶向选择性杀伤能力,并用电镜观察细胞形态。结果重组表达质粒PQE30/EGF-TCS在大肠杆菌M15中获得稳定、高效的融合表达,表达上清柱纯化纯度达95%以上;肝癌BEL-7402细胞表面EGFR表达量最高(72.33%),正常肝LO2细胞表面EGFR表达量最低(5.51%),重组的融合蛋白对肿瘤细胞具有较大的杀伤能力(BEL-7402、MCF-7和BGC-823的IC50分别为11.40、22.47、12.53μg/ml),对正常细胞的杀伤能力微弱(IC50为53.19μg/ml)。结论应用基因工程技术,成功构建了融合蛋白EGF-TCS,该融合蛋白可明显促进肿瘤细胞的凋亡。

Objective To express and purify EGF-TCS fusion protein and observe the targeted and selective killing effect on cancer cells of the fusion protein. Methods The recombinant expression plasmid PQE30/ EGF-TCS was transformed to E. coli. M15 and the fusion protein （EGF-TCS） was expressed. Ni-NTA Agrose affinity chromatography was used to purify the protein, flow cytometry to detect EGFR expression rate in cancer cells （BEL-7402, MCF-7, BGC-823） and normal liver LO2 cells, and the killing test to verify selective killing ability of EGF-TCS; The cell apoptosis detection by flow cytometry and microscopic observation were used to confirm the selective killing ability of EGF-TCS. Results Recombinant expression plasmid PQE30/EGF-TCS was expressed in E. coli. M15 stably and effectively. The purity of EGF-TCS was over 95% by chromatography. EGFR expression rate was highest in hepatoma cells BEL-7402 （72.33%） and lowest in normal liver LO2 cells （5.51% ）. The killing ability of recombinant protein was more effective to cancer cells （ ICs0 of BEL- 7402, MCF-7 and BGC-823 was 11.4, 22.47 and 12.53 μg/ml respectively） and was weak to normal cells （IC50 53.19 μg/ml）. Conclusion The recombinant protein EGF-TCS that induces apoptosis of cancer cells was successfully constructed by gene engineering technology.