Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning
Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning
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DOI：
10.1099/vir.0.80718-0
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发表时间：
2005-04-01
影响因子：
3.8
通讯作者：
Vanderplasschen, A
中科院分区：
文献类型：
--
作者：
Gillet, L;Daix, V;Vanderplasschen, A
牛疱疹病毒4型(BoHV-4)的几个特点使其作为病毒表达载体和/或研究伽马疱疹病毒生物学的模型具有吸引力。然而，由于在真核细胞中使用经典的同源重组来操纵其大基因组的难度，这些发展一直受到阻碍。本研究探索了利用细菌人工染色体(BAC)克隆和原核重组技术生产BoHV-4重组体的可行性。首先，利用两个潜在的插入位点对BoHV-4基因组进行BAC克隆。这两个插入位点都产生了BoHV-4BAC克隆，该克隆稳定地保持在细菌中，并能够在转染到允许细胞中时再生病毒粒子。重组病毒与野生型亲本病毒复制相似，在稳定表达Cre重组酶的允许细胞上生长，切除IoxP侧翼的BAC盒。其次，采用两步诱变的方法，在大肠杆菌中获得了表达蓖麻硬蜱抗补体蛋白I或II(IRAC I/II)的BoHV-4重组体。两个重组子都能诱导感染细胞上清液中高水平的功能IRAC分子的表达。本研究表明，BAC克隆和原核重组技术是发展BoHV-4作为表达载体以及进一步研究这种伽马疱疹病毒的有力工具。
Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the IoxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.