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[目的]构建针对人线粒体转录终止因子2(mitochondrial transcription termination factor 2,MTERF2)的shRNA干扰表达载体,检测其对人宫颈癌HeLa细胞MTERF2基因表达的干扰效率。[方法]以人MTERF2基因为靶向,设计并合成的2条互补寡核苷酸链克隆至pSilencer~(TM)4.1-CMV neo真核表达载体中得到重组干扰质粒。对重组质粒进行PCR和DNA测序鉴定;运用脂质体介导瞬时转染HeLa细胞;采用半定量RT-PCR检测2个干扰质粒对人MTERF2基因mRNA表达的干扰效果,并筛选最有效的shRNA干扰序列;采用Western Blot技术检测2个干扰质粒转染HeLa细胞后MTERF2蛋白的表达情况。[结果]经PCR鉴定和DNA测序鉴定证实,pSi-MTERF2-1和pSi-MTERF2-2重组shRNA干扰表达质粒中已插入目的 DNA片断。瞬时转染MTERF2-shRNA的HeLa细胞48 h后,pSi-MTERF2-1和pSi-MTERF2-2干扰表达质粒均可以显著地降低人MTERF2基因mRNA和蛋白质的表达(P<0.01),而且pSiMTERF2-2干扰效果优于pSi-MTERF2-1。pSi-MTERF2-2对MTERF2基因mRNA和蛋白质表达的抑制率分别为74.0%、78.2%。[结论]成功构建针对人MTERF2基因的shRNA真核表达载体有效抑制目的基因的表达,为进一步应用于宫颈癌的基因治疗研究奠定了基础。
[Objective] To construct shRNA interference expression vector targeting human mitochondrial transcription termination factor 2 (MTERF2), and to detect its interference efficiency on the expression of MTERF2 gene in human cervical cancer HeLa cells. [Method] Two complementary oligonucleotides designed and synthesized targeting human MTERF2 gene were cloned into pSilencer TM 4.1-CMV neo eukaryotic expression vector to obtain recombinant interference plasmids. The recombinant plasmids were identified by PCR and DNA sequencing. HeLa cells were transiently transfected by liposomes. The interference effect of two interfering plasmids on mRNA expression of human MTERF2 gene was detected by semi-quantitative RT-PCR and the most effective shRNA interference The expression of MTERF2 protein in HeLa cells transfected by two interfering plasmids was detected by Western Blot. [Result] The DNA fragments of pSi-MTERF2-1 and pSi-MTERF2-2 were confirmed by PCR identification and DNA sequencing. After transient transfection of HeLa cells transfected with MTERF2-shRNA for 48 h, both pSi-MTERF2-1 and pSi-MTERF2-2 interference expression plasmids significantly reduced the mRNA and protein expression of human MTERF2 (P <0.01), and pSiMTERF2-2 The interference effect is better than pSi-MTERF2-1. The inhibitory rates of pSi-MTERF2-2 on MTERF2 mRNA and protein expression were 74.0% and 78.2%, respectively. [Conclusion] The successful construction of shRNA eukaryotic expression vector targeting human MTERF2 gene effectively inhibits the expression of the target gene, which lays the foundation for the further study of gene therapy for cervical cancer.