Stability of Potato virus X expression vectors is related to insert size:: implications for replication models and risk assessment
Stability of Potato virus X expression vectors is related to insert size:: implications for replication models and risk assessment
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DOI：
10.1007/s11248-006-9051-1
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发表时间：
2007-10-01
影响因子：
3
通讯作者：
Porceddu, Andrea
中科院分区：
文献类型：
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作者：
Avesani, Linda;Marconi, Giampiero;Porceddu, Andrea
我们研究了基于马铃薯X病毒（PVX）的表达构建体的稳定性作为插入片段长度的函数。使用PVX载体在本氏烟草植物中连续传代三次，表达长度范围为261至1，758 bp的五种不同插入片段（人胰岛素原、鼠白细胞介素-10、HIV-1 nef、矮牵牛扩展蛋白-1和人gad 65）。使用竞争性RT-PCR方法，我们证明了插入缺失可能发生在所有插入片段的第一个感染周期，但这更可能是较长的情况。这表明插入片段长度和载体稳定性之间呈负相关。缺失构建体的序列分析表明，重组通常发生在靠近重复的亚基因组启动子的位点，但在少数情况下，可能涉及外源基因本身，导致含有转基因片段的部分缺失构建体。这些结果的背景下，重组蛋白表达及其风险的影响进行了讨论。
We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert-deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.