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Research Article

普及重组杆状病毒来源的OneBac系统用于实验室生产所有重组腺相关病毒载体血清型

卷 21, 期 2, 2021

发表于: 18 January, 2021

页: [167 - 176] 页: 10

弟呕挨: 10.2174/1566523221666210118111657

价格: $65

摘要

背景：重组腺相关病毒（rAAV）已被广泛用作生物医学研究以及基因治疗中的有效转基因载体。与血清型相关的转导效率，组织或细胞类型的嗜性和免疫学特征是rAAV各种应用中的主要考虑因素。对自然分离或人工改造的不同血清型rAAV的需求不断增加。然而，以理想的血清型rAAV的负担得起且可扩展的生产仍然非常困难，特别是对于缺乏相关经验的研究人员而言。 目的：在我们先前建立的源自单个重组杆状病毒表达载体（BEV）的OneBac系统的基础上，我们优化了工艺，并将rAAV的生产范围扩展到了血清型rAAV1-13的整个范围。 方法：首先，通过核糖体渗漏扫描优化AAV Cap基因的翻译，然后将目的基因（GOI）克隆到pFD / Cap-（ITR-GOI）-Rep2穿梭质粒中。遵循经典的Bac-to-Bac方法，可以快速获得包含所有rAAV包装元素的充足BEV库存。最后，我们可以使用单个BEV感染悬浮培养的Sf9细胞，在一周内重复扩大rAAV的产量。 rAAV1-13显示相对较高的产量，范围从5×104到4×105 VG /细胞。从5 L悬浮培养的Sf9细胞中可以轻松获得超过1×1015 VG纯化的rAAV。 结果：正如预期的那样，rAAV血清型1-13对体外转导和细胞型嗜性表现出不同的效力。 结论：总而言之，单个BEV衍生的OneBac系统应被证明可用于实验室放大生产任何血清型rAAV。

关键词: rAAV，重组杆状病毒，OneBac系统，规模化生产，血清型，基因治疗。

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