靶向人核糖核酸酶抑制因子双抗性的 shRNA 逆转录病毒载体构建及鉴定

摘要

目的：构建靶向人核糖核酸酶抑制因子（ hRI）新霉素-潮霉素B双抗性的shRNA逆转录病毒载体，为探讨人核糖核酸酶抑制因子抗肿瘤作用机制提供依据。方法用亚克隆法，将潮霉素B抗性基因hygr从载体pIRES-hygr2克隆到pLNCX-pKD-dsRI-neor与pLNCX-pKD-neor 上，用酶切筛选得到阳性重组质粒pLNCX-pKD-dsRI-neor -hygr与pLNCX-pKD-neor -hygr。转染时设干扰组、空载体组和空白组。用脂质体法将pLNCX-pKD-dsRI-neor （干扰组）、pLNCX-pKD-neor -hygr （空载体组）转染到人肝癌细胞SMMC77细胞中，未处理的细胞作空白组，用400 mg/L G418和200 mg/L潮霉素B筛选3～4周产生稳定的细胞克隆后，用Western blotting检测细胞中核糖核酸酶抑制因子表达的变化。结果酶切结果证明重组质粒构建成功；Western blotting表明，与空白组（0．1810±0．015）和空载体组（0．1951±0．027）相比，干扰组RI表达（0．0311±0．048）明显下调（P＜0．05）。结论成功构建的靶向人核糖核酸酶抑制因子新霉素-潮霉素B双抗性的shRNA逆转录病毒载体能下调RI基因的表达。%Objective To construct the retroviral vector expressing shRNA targeting human ribonuclease inhibitor (hRI) gene carrying both neomycin resistance gene(neor) and hygromycin B resistance gene(hygr)and to investigate its silencing effect on the SMMC7721 cells.Methods The target fragments of hygr from the vectors of pIRES-hygr2 was subcloned into the retroviral vector of pLNCX -pKD-dsRI-neor and its empty vector of pLNCX -pKD-neor respectively.The vector was identified by enzyme digested .And then the shRNA-hRI retroviral vector of pLNCX -pKD-dsRI-neor was trans-fected into SMMC7721 cells with CellfectinR,using the cells transfected with empty vector of pLNCX -pKD-neor and those untransfected as controls .After 3-4 weeks of selection of 400 mg/L G418 and 200 mg/L hygromycin B , the silencing effect of the siRNA plasmid was identified by Western blotting on the SMMC 7721 cells.Results Restriction enzyme diges-tion proved that the construction of the retroviral vector expressing shRNA targeting hRI gene carrying both neor and hygr was correct .Western blotting showed that the expression of hRI were significantly reduced in the cells transfected with shR -NA-hRI retroviral vector as compared with the blank and control group , and the grey scale value of hri/β-actin was (0.0311 ±0.048) vs.(0.1810 ±0.015), (0.1951 ±0.027)(P<0.05).Conclusion The retroviral vector expressing shRNA targeting hRI gene carrying both neor and hygr was successfully constructed and the expression of hRI was effectively inhibitory in the transfected SMMC7721 cells.