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目的探讨构建携带人pre-mir181a基因的重组腺病毒载体,为基因治疗研究提供一种新的方法,并观察其在神经胶质瘤细胞U251细胞中的表达。方法以从Genesil公司购买的含有mir181a的质粒为模板扩增pre-mir181a基因,将回收的PCR产物片段克隆入pGenesil载体,获得重组质粒pGenesil-pre-mir-181a。从pGenesil-pre-mir181a上通过LR体外同源重组将mir181a microRNA表达框转移至pGSadeno腺病毒表达载体上。在体外用不同感染复数(M·O·I)值rAd5-mir181ab分别转染神经胶质瘤细胞U251细胞。采用流式细胞仪、RT-PCR方法检测目的基因的转染效率和表达。结果PCR、酶切鉴定以及序列测定与比对分析表明,重组腺病毒rAd5-mir181a构建正确。神经胶质瘤细胞U251细胞转染rAd5-mir181a的最高效率可达99%,转染后的神经胶质瘤细胞U251细胞表达相应的mir181a。结论本实验成功构建了含mir181a基因的重组腺病毒rAd5-mir181a载体,在体外能高效转染神经胶质瘤细胞U251细胞。
Objective To investigate the construction of a recombinant adenovirus vector carrying human pre-mir181a gene and to provide a new method for gene therapy research and to observe its expression in glioma U251 cells. Methods The plasmid containing mir181a purchased from Genesil Company was used as a template to amplify the pre-mir181a gene. The recovered PCR product fragment was cloned into pGenesil vector to obtain the recombinant plasmid pGenesil-pre-mir-181a. The mir181a microRNA expression cassette was transferred from pGenesil-pre-mir181a by LR in vitro homologous recombination to the pGSadeno adenovirus expression vector. The glioma U251 cells were transfected with rAd5-mir181ab with different MOI values. The transfection efficiency and expression of the target gene were detected by flow cytometry and RT-PCR. Results PCR, restriction enzyme digestion and sequence analysis and comparison analysis showed that the recombinant adenovirus rAd5-mir181a was constructed correctly. Glioma cells U251 cells transfection rAd5-mir181a up to 99% efficiency, the transfected glioma cells U251 cells express the corresponding mir181a. Conclusion The recombinant adenovirus rAd5-mir181a containing mir181a gene was successfully constructed and transfected into glioma U251 cells in vitro.