沉默HepG2细胞核糖核酸酶抑制因子的shRNA逆转录病毒载体的构建及鉴定  

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

作　　者：李琳 李坤[2] 王福强[3] 丁宁[2] 

机构地区：[1]大连市妇幼保健院检验科,辽宁大连116021 [2]大连大学医学院,辽宁大连116021 [3]大连海洋大学,辽宁大连116021

出　　处：《中国医药导报》2014年第14期14-16,27,共4页China Medical Herald

基　　金：辽宁省教育厅科学研究一般项目(编号L2011219);大连大学本科生创新课题(编号2013108)

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。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 subeloned 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.

关 键 词：人核糖核酸酶抑制因子 SHRNA 逆转录病毒载体 构建 

分 类 号：R394.33[医药卫生—医学遗传学]