共沉默Birc5和Hspa5的双干扰siRNA质粒载体构建及鉴定
发布时间：2018-08-21 14:31
【摘要】：目的构建共沉默Birc5和Hspa5的双干扰siRNA质粒,为进一步以Birc5和Hspa5作为肿瘤生物治疗靶点研究提供基础。方法使用Ambion Target Finder设计Birc5和Hspa5mRNA的干扰序列;挑取酶切鉴定正确的质粒转化菌液Birc5-siRNA和Hspa5-siRNA测序鉴定;采用分子克隆技术构建特异性沉默Birc5和Hspa5的双干扰siRNA质粒,命名为pgsiRNA-Birc5+Hspa5,酶切鉴定;将pgsiRNA-Birc5+Hspa5转染HepG2细胞48h后采用Real-time PCR检测Birc5和Hspa5的mRNA表达水平。结果经酶切鉴定正确的质粒转化菌液Birc5-siRNA和Hspa5-siRNA测序结果显示均为插入正确的克隆质粒;经酶切鉴定分析显示pgsiRNA-Birc5+Hspa5符合酶切鉴定结果;转染pgsiRNA-Birc5+Hspa5 48h后HepG2细胞Birc5和Hspa5mRNA表达均显著下降,未转染组Birc5和Hspa5mRNA的相对表达量均为1.0,pgsiRNA-Birc5+Hspa5组Birc5为0.15(P0.05),Hspa5为0.37(P0.05),表明双干扰载体构建成功。结论成功构建了Birc5和Hspa5双干扰siRNA质粒,为进一步同时靶向沉默肿瘤Birc5和Hspa5基因研究提供了工具和基础。
[Abstract]:Objective To construct the co-silenced bi-interfering siRNA plasmids of Birc5 and Hspa5, and to provide the basis for further research on bi-c5 and Hspa5 as targets for tumor biotherapy. Subcloning technique was used to construct the bi-interfering siRNA plasmids with specific silencing of Birc5 and Hspa5, named pgsiRNA-Birc5+Hspa5, which were identified by enzyme digestion. The expression of Birc5 and Hspa5 in HepG2 cells was detected by Real-time PCR 48 hours after pgsiRNA-Birc5+Hspa5 was transfected into HepG2 cells. The results showed that all the cloned plasmids were inserted correctly. The results of enzyme digestion analysis showed that pgsiRNA-Birc5+Hspa5 accorded with the results of enzyme digestion identification. The expression of Birc5 and Hspa5mRNA in HepG2 cells decreased significantly after transfection of pgsiRNA-Birc5+Hspa5 for 48h. The relative expression of Birc5 and Hspa5mRNA in non-transfected group was 1.0, that in pgsiRNA-Birc5+H5 group was 0.15 (P 0.05), and that in Hspa5+H5 group was 0.15 (P 0.05). Spa 5 was 0.37 (P 0.05), indicating that the construction of the double interference vector was successful. Conclusion The double interference siRNA plasmids of Birc 5 and Hspa 5 were successfully constructed, which provided a tool and basis for further study of simultaneous targeted silencing of tumor genes Birc 5 and Hspa 5.
【作者单位】： 武汉科技大学医学院病原生物与免疫学系;杭州医学院病原生物与免疫学教研室;华中科技大学同济医学院基础医学院免疫学系;
【基金】：浙江医学高等专科学校人才引进项目(No.2015B08) 湖北省教育厅科学研究计划重点项目(No.D20131103)
【分类号】：Q78;R73
，
本文编号：2196039
[Abstract]:Objective To construct the co-silenced bi-interfering siRNA plasmids of Birc5 and Hspa5, and to provide the basis for further research on bi-c5 and Hspa5 as targets for tumor biotherapy. Subcloning technique was used to construct the bi-interfering siRNA plasmids with specific silencing of Birc5 and Hspa5, named pgsiRNA-Birc5+Hspa5, which were identified by enzyme digestion. The expression of Birc5 and Hspa5 in HepG2 cells was detected by Real-time PCR 48 hours after pgsiRNA-Birc5+Hspa5 was transfected into HepG2 cells. The results showed that all the cloned plasmids were inserted correctly. The results of enzyme digestion analysis showed that pgsiRNA-Birc5+Hspa5 accorded with the results of enzyme digestion identification. The expression of Birc5 and Hspa5mRNA in HepG2 cells decreased significantly after transfection of pgsiRNA-Birc5+Hspa5 for 48h. The relative expression of Birc5 and Hspa5mRNA in non-transfected group was 1.0, that in pgsiRNA-Birc5+H5 group was 0.15 (P 0.05), and that in Hspa5+H5 group was 0.15 (P 0.05). Spa 5 was 0.37 (P 0.05), indicating that the construction of the double interference vector was successful. Conclusion The double interference siRNA plasmids of Birc 5 and Hspa 5 were successfully constructed, which provided a tool and basis for further study of simultaneous targeted silencing of tumor genes Birc 5 and Hspa 5.
【作者单位】： 武汉科技大学医学院病原生物与免疫学系;杭州医学院病原生物与免疫学教研室;华中科技大学同济医学院基础医学院免疫学系;
【基金】：浙江医学高等专科学校人才引进项目(No.2015B08) 湖北省教育厅科学研究计划重点项目(No.D20131103)
【分类号】：Q78;R73
，
本文编号：2196039
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