共沉默Birc5和Hspa5的双干扰siRNA质粒载体构建及鉴定

摘要

Objective To construct specific siRNA vectors co-silencing the Birc5 and Hspa5 genes,thus providing a basis for researches on tumor biological targeted therapy.Methods Specific siRNA sequences targeting Birc5 and Hspa5 were designed by using Ambion Target Finder.Positive clones were identified by sequencing of Birc5-siRNA and Hspa5-siRNA and named pgsiRNA-Birc5+Hspa5.The expression levels of Birc5 and Hspa5 in HepG2 cells were detected by real-time PCR 48 h after transfection of cells with pgsiRNA-Birc5+Hspa5.Results Birc5-and Hspa5-knockdown siRNA was constructed.The results of restriction enzyme digestion and sequencing were completely correct.The expression levels of Birc5 and Hspa5 mRNA were significantly decreased in HepG2 cells after transfection with pgsiRNA-Birc5+Hspa5.The relative mRNA expression levels of Birc5 and Hspa5 were both 1.0 in the non-transfection group,0.15 and 0.37 in the transfection group(P<0.05).The results indicated that the double interference vector was constructed successfully.Conclusion The Birc5-and Hspa5-knockdown siRNA was constructed successfully,which provides a tool and basis for further study of tumor targeted therapy aimed to Birc5 and Hspa5.%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Hspa5 mRNA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48 h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 48 h后HepG2细胞Birc5和Hspa5 mRNA表达均显著下降,未转染组Birc5和Hspa5 mRNA的相对表达量均为1.0,pgsiRNA-Birc5+Hspa5组Birc5为0.15(P<0.05),Hspa5为0.37(P<0.05),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.