【摘要】 本实验旨在构建人核糖核酸酶抑制因子稳定干涉载体pLKO.1-ds RI,为后续观测人核糖核酸酶抑制因子干涉载体的表达对肿瘤细胞的影响奠定基础。用亚克隆法,将针对人核糖核酸酶抑制因子的干涉片段从Simple T-ds RI质粒克隆到pLK-0.1-TRC质粒,用酶切法筛选得到阳性重组质粒pLKO.1-ds RI,用测序法鉴定克隆序列正确。转染时设干扰组、空载体组和空白组三组,每组三次重复。用脂质体Cellfectin~R将pLKO.1-ds RI(干扰组)、pLKO.1-TRC(空载体组)转染进人肝癌细胞HepG2细胞中,未处理的细胞作空白组。转染后72hr用Western blotting检测细胞中核糖核酸酶抑制因子表达的变化。双酶切鉴定及测序鉴定结果均正确;Western blotting结果表明,对比空白组(1.1578±0.015)和空载体组(1.1216±0.027),干扰组RI表达(0.6119±0.048)明显下调(P<0.05)。结果表明人核糖核酸酶抑制因子干涉载体pLKO.1-ds RNH1构建成功。更多还原

【Abstract】 To construct the interference expression vector of human ribonuclease inhibitor pLKO.1-ds RI which express in tumor cells can lay the foundation for the subsequent observations. The purpose fragments of ds RI from the plasmid of Simple T-ds RI was subcloned into the lentiviral vector of pLKO.1-TRC. The vector was identified by enzyme digested. And then the sh RNA-RI lentiviral vector of pLKO.1-ds RNH1 transfected into HepG2 cells with Cellfectin~R, using the cells transfected with empty vector of pLKO.1-TRC as blank groups and those untransfected cells as controls. After72 hrs, the silencing effect of the siRNA plasmid was identified by Western blotting on the HepG2 cells. Restriction enzyme digestion proved that the construction of the retroviral vector expressing sh RNA targeting h RI gene was correct. Western blotting showed that the expression of h RI were significantly reduced in the cells transfected with sh RNA-h RI retroviral vector as compared with the blank and the empty vector group, and the ratio value of hri/β-actin is 0.6119±0.048 vs 1.1578±0.015, 1.1216±0.027(P<0.05). The human ribonuclease inhibitor interference vector pLKO.1-ds RI was constructed successfully.更多还原