人细胞周期蛋白cyclin D_1基因RNAi表达载体的构建与鉴定

Construction and Identification of Vector Expressing RNA Interference Aimed at Human Cyclin D_1 Gene

[目的]构建针对人细胞周期蛋白cyclinD1基因的RNAi真核表达载体,检测其对人卵巢癌HO-8910细胞cyclinD1基因表达的干扰效率。[方法]以cyclinD1为靶向设计,合成的4条互补寡核苷酸链克隆至pSUPER载体中,得到重组质粒pSUPER-C1￣4,行双酶切分析后测序鉴定;运用脂质体介导转染HO-8910细胞,G418筛选;采用RT-PCR检测对cyclinD1基因mRNA表达的干扰,并筛选有效的siRNA干扰序列。[结果]经酶切测序鉴定证实,重组质粒中已插入目的序列;RT-PCR表明有4个重组载体均不同程度抑制目的基因的转录,但以pSUPER-C2的干扰效率最强。[结论]在卵巢癌细胞系筛选出有效干扰cyclinD1基因表达siRNA序列,成功构建针对cyclinD1基因的RNAi真核表达载体有效抑制目的基因的表达。

[Purpose] To construct the eukaryotic expression vector tot RNA interference human eyelin D1 gene and detect its interference effect in human ovarian cancer cell line （HO-8910）. [Methods] Four target gene segments were synthesized and cloned into pSUPER vector respectively to construct four recombinant eukaryotic expression vectors pSUPER-C1 -4 based on targeted cyclin Dt gene. The four recomhinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then HO-8910 cells were transfected with pSUPER-CI-4 and subjected to G418 selection. In G418-resistant cells, the interference effect was detected by RT-PCR and effective sequence-specific siRNA was screened out. [Resultsl Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSUPER vector. The four recombinant vectors inhibited transc,＇iption of cyclin Dt gene with different extent and pSUPER-C2 had most intensive interference effect. [Conclusion ] The sequence-specific siRNA which could effectively inteffer cyclin D1 gene expression has been screened out. The transcription and expression of cyclin D1 gene are inhibited effectively by the constructed RNAi eukaryotic expression vectors in the ovarian cancer cells.