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表皮生长因子受体由于在许多种肿瘤细胞表面过度表达而成为特异杀伤肿瘤细胞的理想靶位;正电性抗菌肽有独特的膜毒性细胞毒机制。研究以小鼠(Mus musculus)表皮生长因子(mouse epidermal growth factor,MEGF)为导向部分,以蜂毒溶血肽(melittin,Mel)为毒性部分,构建特异杀伤肿瘤细胞的膜毒性免疫毒素MEGFMEL嵌合蛋白。该嵌合蛋白以大肠杆菌(Escherichia coli)BL21为表达宿主,以pET30a为表达载体,采用低温诱导表达和无破胞程序的冻融法进行纯化,最终浓度为63.45μg/mL、纯度为68%。体外活性检测表明,MEGFMEL嵌合蛋白对表面过度表达EGFR的肝癌A431细胞表现出显著杀伤力,其LD50为52.6μg/mL。结果显示以正电性抗菌肽为毒性部分构建针对表皮生长因子受体的新型膜毒性免疫毒素(im-munotoxin,IT)是可行的。
Epidermal growth factor receptor (EGFR) is the ideal target for specific killing of tumor cells because it is overexpressed on the surface of many kinds of tumor cells. Electropositive antibacterial peptides have a unique mechanism of membrane cytotoxicity. In this study, MEGFMEL-chimeric membrane-specific immunotoxin was constructed by targeting murine muscular placental growth factor (MEGF) and melittin (Mel) protein. The chimeric protein was expressed in Escherichia coli BL21 and pET30a was used as the expression vector. The chimeric protein was purified by freeze-thaw method with low-temperature induced expression and non-destructive program. The final concentration was 63.45μg / mL with a purity of 68% . In vitro activity tests showed that the MEGFMEL chimeric protein showed significant cytotoxicity on hepatocellular carcinoma A431 cells overexpressing EGFR on the surface, and its LD50 was 52.6μg / mL. The results showed that it is feasible to construct a novel membrane-bound immunotoxin (EGF) targeting epidermal growth factor receptor by using the electropositive antimicrobial peptide as a toxic moiety.