Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Korea.
Mol Cells. 2009 Jul 31;28(1):19-24. doi: 10.1007/s10059-009-0096-x. Epub 2009 Jul 8.
The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.
本研究的目的是创建一种新型杆状病毒表达系统，该系统不需要进行重组病毒纯化步骤。转染携带 barnase 的转移载体，该基因受小菜蛾颗粒体病毒（CpBV）启动子 ORF3004 或 ORF3005 的控制，会降低细胞生长。与 bApGOZA DNA 共转染不会产生重组病毒和非重组背景。为了进一步研究 barnase 对昆虫细胞的有害影响，构建了两个携带 barnase 基因的重组 bacmid，该基因受 CpBV 启动子的控制，即 bAcFast-3004ProBarnase 和 bAcFast-3005ProBarnase。当仅转染重组 bacmid 时，未观察到病毒复制，但是当 bacmid 与转移载体 pAcUWPolh 共转染时，通过同源重组将 barnase 基因替换为天然多角体蛋白基因，会产生重组病毒。此外，使用 PCR 分析未从未纯化的重组库存中检测到非重组背景。这些结果表明，CpBV 启动子可通过这些启动子控制的致死基因表达来改进杆状病毒表达载体。
