Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.
Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.
复制标题
辅助病毒依赖性腺病毒载体的拯救、繁殖和部分纯化。
DOI：
10.1073/pnas.92.9.3854
复制
发表时间：
1995
影响因子：
11.1
通讯作者：
Kochanek,S
中科院分区：
文献类型：
--
作者：
Mitani,K;Graham,FL;Caskey,CT;Kochanek,S
腺病毒载体作为高效的基因转移载体被广泛应用于包括人类基因治疗在内的多种生物学研究策略中。目前可用的腺病毒载体系统的局限性之一是在载体中存在大部分病毒基因组，导致病毒基因的泄漏表达，特别是在感染的高多重性和外源序列的克隆能力有限。作为克服这一问题的第一步，我们试图修复一个有缺陷的人腺病毒血清5型DNA，该DNA缺失了病毒基因组的一个重要区域（L1, L2, VAI + II, pTP），并被一个指示基因所取代。在野生型腺病毒的帮助下，这种DNA被包装并作为转导病毒颗粒进行繁殖。经过多轮扩增，重组病毒滴度至少达到4 × 10(6)个转导颗粒/ ml。利用CsCl平衡密度梯度离心可从辅助病毒中分离出部分重组病毒。序列繁殖后，重组病毒在标记基因周围的结构保持完整，而在E1区插入的pBR序列被删除。我们的研究结果表明，应该有可能开发一种不编码任何病毒蛋白的辅助腺病毒载体，作为目前可用的腺病毒载体系统的替代方案。
Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.