DNA聚合酶β靶向siRNA真核表达载体的构建和鉴定

Construction and identification of siRNA enkaryotic expression vector targeting for DNA polβ

目的:构建DNA聚合酶β(polβ)靶向的小干扰RNA(siRNA)真核表达载体。方法:根据GenBank中人DNApolβ基因(M13140)的全长序列,利用设计分析软件设计2个DNApolβ靶向发卡样siRNA结构,退火后形成双链,克隆入表达载体pslience2.0-GFP,得到重组质粒pslience2.0-GFP-sipolβ1和pslience2.0-GFP-sipolβ2。通过限制性核酸内切酶酶切和DNA序列测定对重组质粒进行鉴定。结果:经酶切鉴定寡核苷酸已成功插入表达载体pslience2.0-GFP,DNA序列分析表明插入片段序列与合成的siRNA序列相同。结论:成功构建了靶向DNApolβsiRNA真核表达载体。

Aim：To construct the DNA polyinerase β targeting siRNA eukaryotic expression vector.Methods：DNA polβ gene targeted hairpin siRNA was designed according to the whole gene sequence of human DNA polβ gene in Genbank, then four complementary oligonucleotide strands were synthesized and inserted into pslience2.0-GFP vector after annealing to get the new vector pslience2.0-GFP-sipolβ1 and pslience2.0-GFP-sipolβ2.Recombinant plasmid was confirmed with restriction endonuclease digestion and DNA sequencing.Results： Expression vector had been constructed successfully and identified by enzyme digestion analysis. DNA sequence analysis of inserted fragment revealed the same sequences as synthesized siRNA oligonucleotides.Conclusion： DNA polβ siRNA eukaryotic expression vector has been built successfully.