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1013 gene copys/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3 d 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。
Recombinantadeno-associated virus (rAAV) is a developed vector. The rAAV is used to establish animal models of gene over-expression or knock down and as the tool of gene therapy. We use the three plasmids assay to package the rAAV which carries the EGFP gene in HEK293 cells. Through a series of experiments, we establish the method of rAAV purification. In this method, we lyse the cells using sodium deoxycholate, then remove the hybridproteins by adding high concentration NaCl, and purify the rAAV through heparin affinity column. After concentration by ultrafiltration, the rAAV titer can reach to more than 1013 gene copys /mL. The results of different multiplicity of infection (MOI) and time using rAAV to infect HEK 293 cell show that the best condition of infection is MOI=106 and 3 days. After 3 weeks of injecting rAAV to substantia nigra pars compacta of rat midbrain, there are EGFP expressed in the dopaminergic neurons. We have established a method to prepare small amount rAAV in the lab, which can be used in vitro and in vivo.
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