BiZyme: A novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance
BiZyme: A novel fusion protein-mediating selection of vaccinia virus recombinants by fluorescence and antibiotic resistance
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DOI：
10.2144/02325dd08
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发表时间：
2002-05-01
期刊：
影响因子：
2.7
通讯作者：
Hruby, DE
中科院分区：
文献类型：
--
作者：
Hansen, SG;Cope, TA;Hruby, DE
重组牛痘病毒是表达和研究外源基因的有效工具。目前可用于选择牛痘病毒重组体的方法包括病毒空斑形成表型的恢复，在BUdR或霉酚酸存在下病毒DNA的复制，以及在抗生素选择下病毒的成熟和繁殖。虽然有效，但这些方法中的每一种都需要数周的协同努力来分离、纯化和扩增潜在的重组病毒。在这里，我们报告的双功能酶（BiZyme）的发展，以简化和加快分离和纯化的重组牛痘病毒。这种新的选择标记是由编码绿色荧光蛋白和新霉素磷酸转移酶（neo）的基因之间的框内融合组成的。值得注意的是，在6418存在下嵌合gfp-neo盒的表达赋予转染或重组病毒感染的细胞活力和荧光，表明这两种活性都保留在融合蛋白中。因此，将BiZyme掺入重组质粒（pGNR）中以使得能够同时插入感兴趣的外源基因。在这里，我们证明了这种选择/扩增过程需要至少11天来产生所需的牛痘病毒重组体。此外，以这种方式产生的重组体已显示表达生物活性酶和抗原性真实的外源抗原。除了其在牛痘病毒载体系统中的用途之外，BiZyme双功能选择方案应该适用于其他真核和原核表达系统，简单地通过将其与适当的宿主特异性转录调节信号偶联。
Recombinant vaccinia virus is a useful and powerful tool for the expression and study of foreign genes. Methods that are currently available for the selection of vaccinia virus recombinants include the restoration of viral plaque forming phenotype, the replication of viral DNA in the presence of BUdR or mycophenolic acid, and the maturation and propagation of virus under antibiotic selection. Though effective, each of these methods requires several weeks of concerted effort to isolate, purify; and amplify a potential recombinant virus. Here we report the development of a bifunctional enzyme (BiZyme) to simplify and expedite the isolation and purification of vaccinia virus recombinants. This novel selection marker is composed of an in-frame fusion between the genes encoding gfp and the neomycin phosphotransferase enzyme (neo). Remarkably, expression of the chimeric gfp-neo cassette in the presence of 6418 confers both viability and fluorescence to transfected or recombinant virus-infected cells, indicating that both activities are retained within the fusion protein. Therfore, BiZyme was incorporated into a recombination plasmid (pGNR) to enable the concomitant insertion of a foreign gene of interest. Here we demonstrate that this selection/amplification process requires a minimum of 11 days to produce the desired vaccinia virus recombinants. Furthermore, recombinants produced in this fashion have been shown to express both biologically active enzymes and antigenically authentic foreign antigens. In addition to its use in the vaccinia virus vector system, the BiZyme bifunctional selection scheme should be applicable to other eukaryotic and prokaryotic expression systems, simply by coupling it to the appropriate host-specific transcription regulatory signals.