國家衛生研究院 NHRI:Item 3990099045/1142
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    題名: Development of a cell-based assay for monitoring specific hepatitis C virus NS3/4A protease activity in mammalian cells
    作者: Lee, JC;Shih, YF;Hsu, SP;Chang, TY;Chen, LH;Hsu, JTA
    貢獻者: Division of Biotechnology and Pharmaceutical Research
    摘要: The hepatitis C virus (HCV) contains a positive-sense RNA genome that encodes a unique polyprotein precursor, which must be processed by proteases to enable viral maturation. Virally encoded NS3/4A protease has thus become an attractive target for the development of antiviral drugs. To establish an assay system for monitoring NS3/4A protease activity in mammalian cells, this study describes a substrate vector, pEG(Delta4AB)SEAP, in which enhanced green fluorescent protein (EGFP) was fused to secreted alkaline phosphatase (SEAP) through the NS3/4A protease decapeptide recognition sequence, Delta4AB, which spans the NS4A and NS4B junction region. Secretion of SEAP into the culture medium was demonstrated to depend on the cleavage of Delta4AB by HCV NS3/4A protease. We demonstrated that the accumulation of SEAP activity in the culture medium depends on time up to 60 h with the coexpression of active NS3/4A protease. The amount of SEAP in the culture medium was around 10 times greater than that of cells with coexpression of inactive NS3/4A mutant protease. This strategy has made it possible to monitor NS3/4A activity inside mammalian cells. Moreover, by using cells containing the HCV subgenomic replicon, the EG(Delta4AB)SEAP reporter can be used to detect the anti-HCV activity of interferon-alpha (IFN-alpha). Consequently, this EG(Delta4AB)SEAP reporter can be used to screen for NS3/ 4A protease inhibitors in the cellular environment and for anti-HCV drugs in replicon cells. (C) 2003 Elsevier Science (USA). All rights reserved.
    關鍵詞: Biochemical Research Methods;Biochemistry & Molecular Biology;Chemistry, Analytical
    日期: 2003-05-15
    關聯: Analytical Biochemistry. 2003 May;316(2):162-170.
    Link to: http://dx.doi.org/10.1016/S0003-2697(03)00053-8
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0003-2697&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000182606500003
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0037447831
    顯示於類別:[徐祖安] 期刊論文

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