We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH2-terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell-derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus-injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents.

译文

我们从化脓性链球菌基因组中克隆了链球菌溶血素O基因,并测试了将其用作抗癌试剂的可能性。如通过乳酸脱氢酶释放和碘化丙啶摄取所确定的,在转染12小时后,链霉溶血素O基因的瞬时转染有效地杀死了293T细胞。在链球菌溶血素O诱导的细胞死亡期间,未观察到caspase活性,并且坏死明显。链球菌溶血素O蛋白的生化分析表明,对于胆固醇结合活性至关重要的链球菌溶血素O的COOH末端区域仅缺失了5个氨基酸,从而消除了其细胞杀伤活性,而NH2-terminal区域则更具弹性,即,可以在不改变其细胞杀伤活性的情况下删除多达115个氨基酸。我们产生了表达链球菌溶血素O的腺病毒,并将其注射到裸鼠模型中生长的人宫颈癌细胞衍生的肿瘤中。注射后21天,链球菌溶血素O腺病毒注射组的平均肿瘤大小为对照组PBS治疗组的29.3%。我们的结果表明,形成孔的毒素的基因 (如链球菌溶血素O蛋白) 具有建立新型自杀基因治疗试剂的潜力。

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