Quantitative Analysis of the Leaky Expression of Adenovirus Genes in Cells Transduced with a Replication-Incompetent Adenovirus Vector
Quantitative Analysis of the Leaky Expression of Adenovirus Genes in Cells Transduced with a Replication-Incompetent Adenovirus Vector
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DOI：
10.1021/mp200121z
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发表时间：
2011-07-01
影响因子：
4.9
通讯作者：
Mizuguchi, Hiroyuki
中科院分区：
文献类型：
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作者：
Shimizu, Kahori;Sakurai, Fuminori;Mizuguchi, Hiroyuki
从理论上讲，腺病毒(Ad)基因不应该在复制能力不强的Ad载体上表达，因为复制能力不强的Ad载体中缺失了对其他病毒基因表达至关重要的E1a基因。然而，已知的是，病毒基因的泄漏表达发生在E1缺失的Ad载体转导后，导致对Ad蛋白的细胞免疫诱导。到目前为止，还没有对Ad基因的泄漏表达谱进行详细的分析。在这项研究中，我们利用实时定量RT-PCR技术系统地检测了复制不能的重组腺病毒载体(Ad-L2)在感染复数(MOI)分别为10和100的条件下转导细胞后细胞中的Ad基因表达谱，发现在培养细胞中，经Ad-L2转导后的E4和Pix基因有显著的表达。在与野生型Ad相同的MOI下，E4和pix基因的表达水平比转基因(萤火虫荧光素酶)低约30-600倍，比E4和pix基因的表达水平低50-5000倍。出乎意料的是，主要衣壳蛋白的表达水平与背景水平(模拟转导细胞中的Ad基因表达水平)大致相同，甚至略高于背景水平。本研究为设计安全、高效的无复制能力的重组腺病毒载体提供了有价值的信息。
Theoretically, adenovirus (Ad) genes should not be expressed following transduction with a replication-incompetent Ad vector because the E1A gene, which is essential for the expression of other viral gene, is deleted in a replication-incompetent Ad vector. However, leaky expression of viral genes is known to occur following transduction with an E1-deleted Ad vector, leading to an induction of cellular immunity against Ad proteins. To date, no detailed analysis of the leaky expression profiles of Ad genes has been performed. In this study, we systematically examined the expression profiles of Ad genes in cells following transduction with a replication-incompetent Ad vector (Ad-L2) at multiplicities of infection (MOIs) of 10 and 100 using real-time RT-PCR Significant expression was found for the E4 and pIX genes following transduction with Ad-L2 in cultured cells. The expression levels of the E4 and pIX genes were approximately 30- to 600-fold lower than those of the transgene (firefly luciferase), and 50- to 5000-fold lower than those of the E4 and pIX genes following transduction at the same MOI with the wild-type Ad. Unexpectedly, expression levels of the major capsid proteins were approximately the same as, or even slightly above, the background levels (Ad gene expression levels in mock-transduced cells). This study provides valuable information for the design of a safe and efficient replication-incompetent Ad vector.