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。
Objective To obtain the recombinant adeno-associated virus expressing mi RNA-29 b-3 p(r AAV-mi RNA-29 b-3 p), and examine the infection effect and expression level of r AAV-mi RNA-29 b-3 p in the prefrontal cortex of rats. Methods The pre-mi RNA-29 b-3 p 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 LipofiterTMfor r AAV-mi RNA-29 b-3 p packaging. The r AAV-mi RNA-29 b-3 p was harvested from transfected HEK293 cells and purified by a resin column, and the titer of the r AAV-mi RNA-29 b-3 p was tested by q RT-PCR. The r AAV-mi RNA-29 b-3 p was injected to the prefrontal cortex of rats using stereotactic apparatus. The infection effect was observed under immunofluorescence microscope, the expression level of mi RNA-29 b-3 p was detected by q RT-PCR. Results High titer of r AAV-mi RNA-29 b-3 p was obtained after successful packaging. The r AAV-mi RNA-29 b-3 p was purified, the virus titer reached 1.0 ×1012 vg/ml. Strong green fluorescence and high level of mi RNA-29 b-3 p were detected in the infected prefrontal cortex after 21 days of infection. Conclusions The constructed r AAV-mi RNA-29 b-3 p can greatly increase the expression level of mi RNA-29 b-3 p in the prefrontal cortex of rats, which provides a useful tool for further study of the biological function of mi RNA-29 b-3 p.
出处
《中国现代医学杂志》
CAS
北大核心
2017年第25期1-7,共7页
China Journal of Modern Medicine
基金
国家自然科学基金(No:81271478)
