李琳+李坤+王福强+丁宁

    [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。RT-PCR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。

    [关键词] 人核糖核酸酶抑制因子；shRNA；逆转录病毒载体；构建

    [中图分类号] R394.33[文献标识码] A[文章编号] 1673-7210(2014)05(b)-0014-04

    Construction and identification of retroviral vector expressing shRNA targeting hRI gene in HepG2 cell

    LI Lin1 LI Kun2▲ WANG Fuqiang3 DING Ning2

    1.Department of Laboratory Medicine, Maternal and Child Care Service Centre of Dalian City, Liaoning Province, Dalian 116021, China; 2.School of Medicine, Dalian University, Liaoning Province, Dalian 116021，China; 3.Dalian Ocean University, Liaoning Province, Dalian 116021, China

    [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 pKD-dsRI 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. ......