人线粒体转录终止因子3基因RNA干扰表达载体的构建筛选与干扰效果评价

Construction, Identification and Evaluation of RNA Interfenrence Expression Vector Aimed at Human Mitochondrial Transcription Termination Factor 3 Gene

目的:构建人线粒体转录终止因子3(MTERF3)基因的短发夹RNA(shRNA)干扰表达载体,并在mRNA和蛋白质水平对其干扰效率进行验证,以检测和筛选出干扰效率最优的shRNA表达载体。方法:根据人MTERF3基因全长cDNA序列,利用Oligoengine在线软件设计4个shRNA序列,合成4条互补寡核苷酸链,退火成双链后克隆至psiU6.1载体,得到4个重组干扰质粒psi-MTERF3-1～psi-MTERF3-4,并通过PCR和DNA测序对其进行鉴定;将重组干扰质粒利用脂质体介导瞬时转染He La细胞,转染48 h后采用real time RT-PCR检测4个干扰质粒对MTERF3 mRNA表达水平的影响,采用Western印迹检测其对MTERF3蛋白表达水平的情况,并筛选出最有效的shRNA干扰质粒。结果:PCR鉴定和DNA测序鉴定证实重组质粒中已插入目的DNA序列;real time RT-PCR和Western印迹结果表明4个重组载体均可以显著降低人MTERF3基因mRNA和蛋白质的表达水平(P<0.05),其中以pSi-MTERF3-4的干扰效率最优,对MTERF3 mRNA表达的抑制率为70.1%,对MTERF3蛋白表达的抑制率为72.2%。结论:在人宫颈癌He La细胞系筛选出有效干扰MTERF3基因表达的shRNA序列,构建了针对MTERF3基因的RNA干扰真核表达载体且能够有效抑制目的基因的表达,为进一步研究人MTERF3在宫颈癌发生发展中的作用机制提供了实验基础。

Objective: To construct the eukaryotic expression vector for RNA interference human mitochondrial transcription termination factor 3(MTERF3) gene, and verify the effect of interference at the level of m RNA and protein expression, so as to choose a optimal interference vector. Methods: Four pairs of sh RNA oligonucleotide sequences were designed and synthesized according to human MTERF3 gene sequence in Gen Bank(NM_015942.4), and then were annealed into double-strand and inserted into plasmid of psi-U6.1 respectively to contruct expression vectors psi-MTERF3-1～psi-MTERF3-4. The four recombinant vectors were identified by PCR and DNA sequencing. Then He La cells were transfected with psi-MTERF3-1～psi-MTERF3-4. MTERF3 m RNA expression and protein expression were detected by real time RT-PCR and Western blot, and then effective sequencespecific si RNA was screened out. Results: PCR and DNA sequencing showed that the target segments were cloned into psi-U6.1 vector. The four recombinant vectors inhibited transcription of MTERF3 gene with different extent and psi-MTERF3-2 had most intensive interference effect. Conclusion: The sequence-specific si RNA which could effectively interfer MTERF3 gene expression has been screened out. The transcription and expression of MTERF3 gene are inhibited effectively by the constructed RNAi eukaryotic expression vectors in the human cervical cancer He La cell line. This study provides an experimental basis for further study on the role of MTERF3 in development of human cervical cancer.