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

Construction of recombinant adeno-associated virus expressing miRNA-29b-3p and its expression in prefrontal cortex of rats

摘要:

目的获得表达microRNA-29b-3p 的重组腺相关病毒（rAAV-miR-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miR-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LipaFiterTM共转染HEK293 细胞包装rAAVmiR-29b-3p。从转染的HEK293 细胞中收集并通过树脂柱纯化rAAV-miR-29b-3p，通过实时荧光定量聚合酶链反应（qRT-PCR）测定rAAV-miR-29b-3p滴度。通过脑立体定位仪注射rAAV-miR-29b-3p 至大鼠前额叶皮质，免疫荧光显微镜观察rAAV-miR-29b-3p的感染效果，qRT-PCR检测rAAV-miR-29b-3p表达水平。结果成功包装的rAAV-miR-29b-3p 经纯化后获得了高纯度的rAAV-miR-29b-3p，qRT-PCR检测rAAV-miR-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，miR-29b-3p 表达水平明显提高。结论构建的rAAV-miR-29b-3p 可使大鼠前脑皮层miR-29b-3p 高表达，为进一步研究miR-29b-3p的生物学功能提供了有效工具。

Abstract:

Objective To obtain the recombinant adeno-associated virus expressing miRNA-29b-3p (rAAVmiRNA-29b-3p), and examine the infection effect and expression level of rAAV-miRNA-29b-3p in the prefrontal cortex of rats. Methods The pre-miRNA-29b-3p gene fragment was inserted into AAV expression plasmid to construct a recombinant AAV plasmid. The recombinant AAV plasmid, packaging plasmid and helper plasmid were co-transfected into HEK293 cells by LipofiterTM for rAAV-miRNA-29b-3p packaging. The rAAV-miRNA-29b-3p was harvested from transfected HEK293 cells and purified by a resin column, and the titer of the rAAV-miRNA-29b-3p was tested by qRT-PCR. The rAAV-miRNA-29b-3p was injected to the prefrontal cortex of rats using stereotactic apparatus. The infection effect was observed under immunofluorescence microscope, the expression level of miRNA-29b-3p was detected by qRT-PCR. Results High titer of rAAV-miRNA-29b-3p was obtained after successful packaging. The rAAV-miRNA-29b-3p was purified, the virus titer reached 1.0×1012 vg/ml. Strong green fluorescence and high level of miRNA-29b-3p were detected in the infected prefrontal cortex after 21 days of infection. Conclusions The constructed rAAV-miRNA-29b-3p can greatly increase the expression level of miRNA-29b-3p in the prefrontal cortex of rats, which provides a useful tool for further study of the biological function of miRNA-29b-3p.