【摘要】 目的构建针对人核糖核酸酶抑制因子(hRI)的shRNA逆转录病毒载体,为探讨hRI抗肿瘤作用机制打下基础。方法用亚克隆法将目的片段pkd-dsRI和pkd从表达载体pKD-dsRI克隆到pLNCX上,用双酶切筛选得到阳性克隆后,用脂质体法将其转染到人肝癌细胞HepG2细胞中,用800 mg/L G418筛选2周,产生稳定的细胞克隆后,用RT-PCR检测细胞中核糖核酸酶抑制因子mRNA表达的变化。结果双酶切鉴定为阳性克隆。RTPCR表明,对比空白组(0.790±0.014)和空载体组(0.904±0.027),干扰组hri基因表达(0.361±0.048)明显下调,差异有统计学意义(P<0.05)。结论成功构建了针对hRI的shRNA逆转录病毒载体。更多还原

【Abstract】 Objective To construct the retroviral vector expressing shRNA targeting hRI gene, in order to provide basis for discussion of hRI antitumor effect. Methods The target fragments of pkd-dsRI and pkd from the vectors of pKDdsRI were subcloned into the retroviral vector pLNCX respectively. The vector was identified by enzyme digested, then transfected into HepG2 cells with liposome method. After 2 weeks of selection of 800 mg/L G418, the silencing effect of the siRNA plasmid was identified by RT-PCR on the HepG2 cells. Results Restriction enzyme digestion proved that the construction of retroviral vector expressing shRNA targeting hRI gene was correct. RT-PCR showed that the expression of hri gene were significantly reduced in the HepG2 cells transfected with shRNA-hRI retroviral vector(0.361±0.048) as compared with the blank control group(0.790±0.014) and the blank retroviral vector group(0.904± 0.027), with statistically significant difference(P < 0.05). Conclusion The retroviral vector expressing shRNA targeting hRI gene is successfully constructed.更多还原