Hansen Scott G, Cope Torrey A, Hruby Dennis E
Oregon State University, Corvallis, OR 97331, USA.
Biotechniques. 2002 May;32(5):1178, 1180, 1182-7. doi: 10.2144/02325dd08.
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 G418 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 authenticforeign 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.
重组痘苗病毒是用于外源基因表达和研究的一种有用且强大的工具。目前可用于选择痘苗病毒重组体的方法包括病毒蚀斑形成表型的恢复、在存在溴脱氧尿苷（BUdR）或霉酚酸的情况下病毒DNA的复制，以及在抗生素选择下病毒的成熟和增殖。尽管这些方法有效，但每种方法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。在此我们报告了一种双功能酶（BiZyme）的开发，以简化和加速痘苗病毒重组体的分离与纯化。这种新型选择标记由编码绿色荧光蛋白（gfp）的基因与新霉素磷酸转移酶（neo）的读框内融合组成。值得注意的是，在存在G418的情况下嵌合gfp-neo盒的表达赋予转染或重组病毒感染细胞活力和荧光，表明两种活性都保留在融合蛋白中。因此，BiZyme被整合到一个重组质粒（pGNR）中，以便能够同时插入感兴趣的外源基因。在此我们证明，这个选择/扩增过程最少需要11天来产生所需的痘苗病毒重组体。此外，以这种方式产生的重组体已被证明能表达具有生物活性的酶和抗原性真实的外源抗原。除了用于痘苗病毒载体系统外，BiZyme双功能选择方案只需将其与适当的宿主特异性转录调控信号偶联，就应该适用于其他真核和原核表达系统。
