MicroRNA-29b-3p重组腺相关病毒制备及在大鼠前额叶皮质中的表达研究

发布时间：2018-05-07 17:24

本文选题：腺相关病毒 + mi　；参考：《中国现代医学杂志》2017年25期

【摘要】：目的获得表达micro RNA-29b-3p的重组腺相关病毒(rAAV-mi R-29b-3p),并检测其在大鼠前额叶皮质中的感染效果和表达水平。方法将前体mi R-29b-3p基因片段置入AAV表达质粒中构建重组AAV质粒,将重组AAV质粒、包装质粒和辅助质粒通过脂质体Lipa Fiter TM共转染HEK293细胞包装r AAVmi R-29b-3p。从转染的HEK293细胞中收集并通过树脂柱纯化r AAV-mi R-29b-3p,通过实时荧光定量聚合酶链反应(q RT-PCR)测定r AAV-mi R-29b-3p滴度。通过脑立体定位仪注射r AAV-mi R-29b-3p至大鼠前额叶皮质,免疫荧光显微镜观察r AAV-mi R-29b-3p的感染效果,q RT-PCR检测r AAV-mi R-29b-3p表达水平。结果成功包装的r AAV-mi R-29b-3p经纯化后获得了高纯度的r AAV-mi R-29b-3p,q RT-PCR检测r AAV-mi R-29b-3p的病毒滴度达到1.0×1012 vg/ml,经感染21 d后,脑组织前额叶皮质荧光表达明显,mi R-29b-3p表达水平明显提高。结论构建的r AAV-mi R-29b-3p可使大鼠前脑皮层mi R-29b-3p高表达,为进一步研究mi R-29b-3p的生物学功能提供了有效工具。
[Abstract]:Objective to obtain recombinant adeno-associated virus (rAAV-mi R-29b-3pN) expressing micro RNA-29b-3p, and to detect its infection effect and expression level in prefrontal cortex of rats. Methods the precursor mi R-29b-3p gene fragment was inserted into the AAV expression plasmid to construct the recombinant AAV plasmid. The recombinant AAV plasmid, the packaging plasmid and the auxiliary plasmid were co-transfected into HEK293 cell package r AAVmi R-29b-3p. by liposome Lipa Fiter TM. R AAV-mi R-29b-3pwas collected from transfected HEK293 cells and purified by resin column. The titer of r AAV-mi R-29b-3p was determined by real-time quantitative polymerase chain reaction (Q RT-PCR). R AAV-mi R-29b-3p was injected into the prefrontal cortex of rats by stereotactic locator. The infection effect of r AAV-mi R-29b-3p was observed by immunofluorescence microscope and the expression of r AAV-mi R-29b-3p was detected by Q RT-PCR. Results the virus titer of r AAV-mi R-29b-3p detected by highly purified r AAV-mi R-29b-3pnq RT-PCR was 1. 0 脳 1012 vg / ml. After 21 days of infection, the fluorescence expression of R-29b-3p in prefrontal cortex of brain tissue was significantly increased. Conclusion the constructed r AAV-mi R-29b-3p can increase the expression of mi R-29b-3p in rat forebrain cortex, which provides an effective tool for further study of the biological function of mi R-29b-3p.
【作者单位】：西南医科大学附属医院麻醉科;
【基金】：国家自然科学基金(No:81271478)
【分类号】：R3416

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