突变减毒志贺样毒素Ⅰ真核表达载体的构建及其抗卵巢癌的活性

Construction of recombinant eukaryotic expression vectors carrying genes encoding attenuated Shiga-like toxin Ⅰ mutant and their anti-oophoroma effect

目的构建表达突变减毒的志贺样毒素Ⅰ(Shiga-liketoxin1,Stx1)的真核表达载体,并考察其抗人卵巢癌细胞SKOV3的活性。方法重叠PCR法构建毒性为原毒素毒性1/10和1/100的突变减毒Stx1编码序列,T-A克隆并测序后分别连入真核表达载体pcDNA3.1。转染SKOV3细胞,RT-PCR法检测Stx1mRNA在SKOV3细胞中的表达,观察突变减毒Stx1对SKOV3细胞周期的影响和致SKOV3细胞死亡的方式。使用SKOV3荷瘤裸鼠模型,不同毒性突变减毒Stx1真核表达载体经脂质体包裹后肿瘤局部注射给药,评价其体内抑瘤能力。结果突变减毒Stx1真核表达载体经酶切和测序鉴定与预期突变序列一致;RT-PCR证实转染后的SKOV3细胞中存在目的mRNA;体外实验观察到该载体转染可以使人卵巢癌SKOV3细胞的细胞周期阻滞于G2/M期,转染后SKOV3细胞死亡的主要途径是坏死;体内实验显示突变减毒Stx1真核表达载体可以抑制裸鼠体内SKOV3移植瘤的生长。结论通过重叠PCR方法获得2种突变减毒Stx1编码序列,成功构建了相应的真核表达载体,并证实该载体具有明显的抗卵巢癌活性。

Objective： To construct recombinant eukaryotic expression vectors carrying genes encoding attenuated Shiga-like toxin 1 mutant and to study their anti-oophoroma effect in vitro and in vivo. Methods： The genes encoding attenuated Shiga-like toxin 1 mutant were amplified by overlap PCR and then cloned into eukaryotic expression plasmid pcDNA3. 1. The recombinant plasmids were transfected into SKOV3 cells and RT-PCR was used determine the expression of Stx 1 mRNA in SKOV3 cells. The influence of attenuated Stx 1 on the cell cycle of SKOV3 cells and the mechanism by which Stx 1 induces apoptosis of SKOV3 cells were observed. Then Stx 1 with different cyotoxicites （ after packed by liposome） were intratumorally injected into immunodeficient mice harboring SKOV3 to assess their anti-oophoroma effect in vi- vo. Results： It was showed that the genes encoding attenuated Stx 1 mutant were successfully cloned into pcDNA3.1 and their mRNA expression in transfected SKOV3 cells was verified by RT-PCR. In vitro study showed that the constructed plasmid arrested SKOV3cells at G2/M stage. The post-transfection death of SKOV3 cells was mainly through necrosis as assessed by flow cytometry. In vivo study showed that the constructed plasmid had a significant anti-oophoroma effect on the growth of SKOV3 tumor in immunodefficient mice. Conclusion： Two sequences encoding attenuated Stx 1 mutant have been successfully constructed and expressed in eukaryotic expression system, which have been confirmed to have obvious anti-oophoroma effect in vitro and in vivo.