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科技导报  2015 

重组腺相关病毒的小量制备与体内体外感染研究

DOI: 10.3981/j.issn.1000-7857.2015.18.012, PP. 72-77

Keywords: 重组腺相关病毒,病毒包装,病毒纯化

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Abstract:

重组腺相关病毒(rAAV)是近年来发展的较为成熟的一种病毒基因载体,常用于过表达或者敲低等动物模型的建立与基因治疗等。本研究使用三质粒共转染的方法,在HEK293细胞中包装出含绿色荧光蛋白(EGFP)基因的rAAV,通过一系列实验,确定纯化方法为脱氧胆酸钠裂解,高浓度NaCl去除杂蛋白,最后通过肝素层析柱纯化,经超滤管浓缩后其滴度可达1013genecopys/mL以上。将纯化后的rAAV感染HEK293细胞,通过实验确定使用感染复数为106、感染3d的细胞能够表达出高水平的EGFP。将rAAV注射入大鼠中脑黑质致密部,经过3周的感染发现,rAAV可以特异性地感染多巴胺能神经元,表达出EGFP。通过以上实验,建立了一个在实验室小量制备rAAV的方法,且此方法制备的rAAV完全满足体内与体外实验的要求。

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