rAAV-NGF-GFP载体的构建及鉴定

Construction and identification of rAAV-NGF-GFP expression vector

目的:构建携带神经生长因子(nerve growth factor,NGF)的重组腺相关病毒载体(recombinant deno-associated virus,rAAV),为临床利用神经生长因子治疗阿尔兹海默病(Alzheimer’sdisease,AD)提供实验依据。方法:采用基因重组技术构建含有NGF基因的AAV-NGF载体,将pAAV-NGF-GFP、pRC、pHelper 3种质粒共转染HEK293细胞,包装获取重组腺相关病毒,采用SDS-PAGE胶和Dot-blot检测病毒纯度和滴度;重组腺相关病毒转染人类神经母细胞瘤细胞(SK),荧光显微镜下观察GFP表达。结果:成功构建rAAV-NGF-GFP载体,滴度为1.7×1010v.g/ml,将病毒转染SK细胞48 h后细胞中有荧光表达。结论 :构建的rAAV-NGF-GFP病毒载体可成功转染SK细胞。

Objective：To construct rAAV-NGF-GFP, a recombinant adeno-associated virus-based gene delivery vector in order to provide experimental basis for clinical AD treatment by NGF. Methods： AAV-NGF delivery vector containing NGF gene was constructed by gene recombination technology, the recombinant AAV-NGF-GFP was produced by calcium phosphate precipitate, which was formed by mixing pAAV-RC, pHelper and pAAV-NGF contransfected into human embryonic kidney （HEK） 293 cells. The rAAV-NGF-GFP was purified and condensed. The purity and the titer of rAAV-NGF-GFP was tested by SDS-PAGE and DNA dot-blot respectively. The GFP transfected by rAAV-NGF-GFP was observed under the inverted fluorescence microscope. Results： The expressing vector of rAAV-NGF-GFP was sucessfully constructed titer of rAAV-NGF-GFP was 1.7 × 1010 v.g/ml. Green fluorescent cells could be observed under the inverted fluorescent microscope at 48 hours after transfecting the recombinant plasmid DNA vector into SK. Conclusion： The recombinant expression vector rAAV-NGF-GFP can be successfully transfected into SK cells.