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目的:表达并纯化转化生长因子α与绿脓杆菌外毒素融合蛋白(TGFα-PE40),探讨其对EGF受体(EGFR)阳性肿 瘤细胞的靶向杀伤作用。方法:用pET28a表达载体构建表达TGFα-PE40蛋白的重组体pV28,IPTG诱导其表达后,提取包涵 体蛋白并用Ni柱纯化,MTT法观察复性融合蛋白对肿瘤细胞A431和SK-OV3的杀伤效用。结果:成功构建基因重组质粒 pV28,纯化后的包涵体蛋白中TGFα-PE40蛋白纯度达98％以上,这种活性毒素蛋白对EGFR高表达的A431癌细胞抑制率为 50％(IC50)时所需蛋白浓度为(0.86±0.07)μg／ml,低于EGFR低表达的SK-OV3癌细胞的IC50(6.37±2.18μg/ml),差异显 著(P<0.05)。结论:融合蛋白TGFα-PE40对肿瘤细胞的毒性与肿瘤细胞表面表达的EGFR数量成正相关,可选择性杀伤肿 瘤细胞。
OBJECTIVE: To express and purify transforming growth factor-α (TGF-α) and Pseudomonas aeruginosa exotoxin fusion protein (TGFα-PE40) and to investigate its targeted killing effect on EGFR positive tumor cells. METHODS: Recombinant pV28 expressing TGFα-PE40 protein was constructed by pET28a expression vector. After induced by IPTG, the inclusion body protein was extracted and purified by Ni column. The killing effect of the fusion protein on A431 and SK-OV3 tumor cells was observed by MTT assay . Results: The recombinant plasmid pV28 was successfully constructed. The purity of the purified TGFα-PE40 protein was over 98%. The inhibitory rate of the active toxin protein to the highly expressed A431 cancer cells with EGFR was 50% (IC50) The protein concentration was (0.86 ± 0.07) μg / ml, which was significantly lower than that of SK-OV3 cells with low EGFR expression (6.37 ± 2.18μg / ml) (P <0.05). Conclusion: The cytotoxicity of fusion protein TGFα-PE40 to tumor cells is positively correlated with the number of EGFR expressed on the surface of tumor cells, which can selectively kill tumor cells.