Chen Ying-Ju, Chen Wein-Shue, Wu Tzong-Yuan
Department of Bioscience Technology, Chung Yuan Christian University, Chungli 320, Taiwan, ROC.
Biochem Biophys Res Commun. 2005 Sep 23;335(2):616-23. doi: 10.1016/j.bbrc.2005.07.116.
A bi-cistronic baculovirus transfer vector was constructed based on the 5'UTR internal ribosome entry site (IRES) of the Rhopalosiphum padi virus (RhPV). Recombinant baculoviruses containing the red fluorescent protein gene and green fluorescent protein gene flanking the RhPV 5'UTR IRES can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. Quantization by fluorescence spectrophotometry of the fluorescent proteins produced in Sf21 cells indicated that the translational efficacy of the RhPV 5'UTR IRES was about 3-fold weaker than cap-dependent translation. We also demonstrated that recombinant baculoviruses containing the human interferon-gamma gene (IFN-gamma) and green fluorescent protein gene flanking the RhPV 5'UTR IRES can produce IFN-gamma proteins as well as green fluorescent proteins. These results suggest that the RhPV IRES can be used in the development of bi-cistronic baculovirus expression vectors for production of heterologous multiprotein complexes or can be combined with selection markers to facilitate applications of baculovirus expression systems.
基于麦二叉蚜病毒（RhPV）的5'非翻译区内部核糖体进入位点（IRES）构建了一种双顺反子杆状病毒转移载体。含有位于RhPV 5'UTR IRES两侧的红色荧光蛋白基因和绿色荧光蛋白基因的重组杆状病毒，在多角体蛋白启动子的控制下，可在重组病毒感染的草地贪夜蛾21细胞（Sf21）中同时产生双荧光。通过荧光分光光度法对Sf21细胞中产生的荧光蛋白进行定量分析表明，RhPV 5'UTR IRES的翻译效率比帽依赖性翻译弱约3倍。我们还证明，含有位于RhPV 5'UTR IRES两侧的人γ干扰素基因（IFN-γ）和绿色荧光蛋白基因的重组杆状病毒能够产生IFN-γ蛋白以及绿色荧光蛋白。这些结果表明，RhPV IRES可用于开发双顺反子杆状病毒表达载体以生产异源多蛋白复合物，或者可与选择标记结合以促进杆状病毒表达系统的应用。
