杆状病毒表达系统制备的重组禽腺联病毒的鉴定
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摘要
为对杆状病毒表达系统制备的重组禽腺联病毒进行生物学特性的鉴定,将含GFP报告基因及AAAV两侧末端重复序列的重组杆状病毒rBac-GFP、表达AAAV结构蛋白的重组杆状病毒rBac-VP、表达AAAV功能蛋白的重组杆状病毒rBac-Rep以感染复数为5,同时感染摇瓶培养中的昆虫细胞Sf9,72 h后收集细胞沉淀,反复冻融3～5次后,离心取上清。经滤膜过滤、氯仿抽提和PEG沉淀后,进行电镜观察和Western blot分析,并体外感染鸡成纤维细胞和鸡肝细胞系。SDS-PAGE结果显示,rAAAV得到了较好的纯化,电镜下可观察到大小约20 nm的典型细小病毒样粒子,Western blot分析数据显示rAAAV由3个结构蛋白组成,与野生病毒相似。体外表达试验结果显示,rAAAV能介导GFP报告基因在鸡细胞中稳定持久地表达。表明杆状病毒表达系统制备的rAAAV具有与野生病毒相似的性质,可应用于后继研究和开发应用。
引文
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[14]Lock M,Alvira M,Vandenberghe L H,et al. Rapid,simple,and versatile manufacturing of recombinant Adeno-Associated viral vectors at scale[J]. Human Gene Therapy,2010,21(10):1259-1271.
[2]Koeberl D D,Alexander I E,Halbert C L,et al. Persistent expression of human clotting factor Ⅸ from mouse liver after intravenous injection of adeno-associated virus vectors[J]. Proceedings of the National Academy of Sciences of the United States of America,1997,94:1426-1431.
[3]Afione S A,Conrad C K,Kearns W G,et al. In vivo model of adeno-associated virus persistence and rescue[J]. Journal of Virology,1996,70:3235-3241.
[4]Harrison P T,Dalziel R G,Ditchfield N A,et al. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector[J]. Neuroscience,1999,94:997-1003.
[5]Rolling F,Shen W Y,Tabarias H,et al. Evaluation of adeno-associated virus-mediated gene transfer into the rat retina by clinical fluorescence photography[J]. Human Gene Therapy,1999,10(4):641-648.
[6]Xiao X A,Li J A,Samulski R J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector[J]. Journal of Virology,1996,70(11):8098-8108.
[7]Yates V J,El-Mishad A M,McCormick K J,et al. Isolation and characterization of an avian adenovirus-associated virus[J]. Infec Immun,1973(7):973-980.
[8]Bossis I,Chiorin J A. Cloning of an avian adeno-associated virus(AAAV)and generation of recombinant AAAV particles[J]. Journal of Virology,2003,77(12):6799-6810.
[9]Estevez C,Villegas P. Sequence analysis,viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus[J]. Virus Research,2004,105(2):195-208.
[10]Urabe M,Ding C T,Kotin R M. Insect cells as a factory to produce adeno-associated virus type 2 vectors[J]. Human Gene Therapy,2002,13:1935-1943.
[11]Wang A P,Wang Y J,Wu S,et al. Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system[J]. Journal of Virological Methods,2017,240:26-31.
[12]Farson D,Harding T C,Tao L,et al. Development and characterization of a cell line for large-scale,serum-free production of recombinant adeno-associated viral vectors[J]. Journal of Gene Medicine,2004(6):1369-1381.
[13]Durocher Y,Pham P L,St-Laurent G,et al. Scalable serum-free production of recombinant adeno-associated virus type 2 by transfection of 293 suspension cells[J]. Journal of Virological Methods,2007,144(1/2):32-40.
[14]Lock M,Alvira M,Vandenberghe L H,et al. Rapid,simple,and versatile manufacturing of recombinant Adeno-Associated viral vectors at scale[J]. Human Gene Therapy,2010,21(10):1259-1271.