期刊
ANTIVIRAL RESEARCH
卷 87, 期 1, 页码 50-56出版社
ELSEVIER
DOI: 10.1016/j.antiviral.2010.04.010
关键词
NS3/4A protease; Split firefly luciferase complementation strategy; In vivo imaging
资金
- Natural Science Foundation of China [30600330, 30671842, 30672488, 30700475, 30771919, 30700757]
- National High Technology Research and Development Program of China [2008AA02Z132]
- Beijing, Municipal Natural Science Foundation [5082016]
- Mega-projects of Science Research for the 11th Five-Year Plan [2009ZX10004-4001]
The lack of robust small animal models has been an obstacle to the screening of Hepatitis C virus (HCV) NS3/4A protease inhibitors in vivo. Here, we described a reporter assay system for in vivo noninvasive imaging of NS3/4A serine protease activity using split firefly luciferase complementation strategy. The reporter construct ANIuc(NS5A/B)BCluc constitutes the split N- and C-terminal fragments of luciferase, fused to interacting peptides, pepA and pepB, respectively, with an intervening HCV NS3/4A cleavage motif of NSSA/B. we proved that the reporter molecule could be proteolytically cleaved by NS3/4A at the NS5A/B motif in cells and living animals. Association of pepA and pepB brought inactive fragments of luciferase into close proximity, thereby restoring bioluminescence activity. The increase in luciferase activity was proportional to the dose of active NS3/4A protease. The ANIuc(NS5A/B)BCluc reporter also could be used to detect the activity of NS3/4A-specific shRNA and IFN-alpha. Therefore, the reporter assay system using split firefly luciferase complementation strategy should prove useful for evaluating NS3/4A protease activity in cells and living animals. (C) 2010 Elsevier B.V. All rights reserved.
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