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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 an eukaryotic expression vector expressing attenuated Shiga-liketoxin 1 (Stx1) and investigate its anti-human ovarian cancer cell line SKOV3. Methods The overlapped PCR method was used to construct the mutated and attenuated Stx1 coding sequence of virulence virulence 1/10 and 1/100. The T-A clones were cloned and sequenced and then ligated into the eukaryotic expression vector pcDNA3.1. The expression of Stx1 mRNA in SKOV3 cells was detected by RT-PCR. The effect of attenuated Stx1 on the cell cycle of SKOV3 cells and the death of SKOV3 cells were observed. Using SKOV3 tumor-bearing nude mouse model, different expression levels of Stx1 eukaryotic expression vector were encapsulated by liposomes and injected into the tumor locally to evaluate the antitumor activity. Results The mutated and attenuated Stx1 eukaryotic expression vector was identified by restriction enzyme digestion and sequencing. The target gene was identified by RT-PCR and the transfected SKOV3 cells were confirmed by RT-PCR. The transfected SKOV3 cells were transfected with human ovarian cancer SKOV3 The cell cycle arrest in G2 / M phase. The main pathway of SKOV3 cell death after transfection was necrosis. In vivo experiments showed that the mutant attenuated Stx1 eukaryotic expression vector can inhibit the growth of SKOV3 xenografts in nude mice. Conclusion Two kinds of mutated and attenuated Stx1 coding sequences were obtained by overlapping PCR. The corresponding eukaryotic expression vector was successfully constructed and proved to have obvious anti-ovarian cancer activity.