國家衛生研究院 NHRI:Item 3990099045/1149
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    题名: A reporter-based assay for identifying hepatitis C virus inhibitors based on subgenornic replicon cells
    作者: Lee, JC;Chang, CF;Chi, YH;Hwang, DR;Hsu, JTA
    贡献者: Division of Biotechnology and Pharmaceutical Research
    摘要: Hepatitis C virus (HCV) encodes a polyprotein that needs to be processed proteolytically by cellular and viral proteases into mature functional proteins. One of the viral proteins, NS3/4A, has serine protease activity that is critical for virus maturation. The generation and characterization of an engineered HCV replicon cell line (Ava5) is described which constitutively expresses EG(Delta4AB)SEAP reporter protein and the cell line was designated as Ava5-EG(Delta4AB)SEAP.EG(Delta4AB)SEAP is a fusion protein 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. The secretion of SEAP into culture medium has been shown to depend on the cleavage of Delta4AB by HCV NS3/4A protease. It is demonstrated that the amount of NS3/4A in Ava5-EG(Delta4AB)SEAP cells correlated well with the copy numbers of HCV subgenomic RNA. It is also shown that replication of HCV subgenomic RNA inside cells is reflected by the alkaline phosphatase (SEAP) levels in culture medium. SEAP activity in the culture medium of Ava5-EG(Delta4AB)SEAP was approximately 50-fold higher than the parental Ava5 cells. Ava5-EG(Delta4AB)SEAP was validated as a drug screening system since several known HCV inhibitors were shown to reduce SEAP activities in culture media of Ava5-EG(Delta4AB)SEAP cells. In conclusion, Ava5-EG(Delta4AB)SEAP cells can be used to monitor HCV sub-genomic replication and the assay can be readily adapted to high throughput screening format to identify prospective anti-HCV drugs. (C) 2003 Elsevier B.V. All rights reserved.
    关键词: Biochemical Research Methods;Biotechnology & Applied Microbiology;Virology
    日期: 2004-03-01
    關聯: Journal of Virological Methods. 2004 Mar;116(1):27-33.
    Link to: http://dx.doi.org/10.1016/j.jviromet.2003.10.007
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0166-0934&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000188380600004
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0346096567
    显示于类别:[徐祖安] 期刊論文
    [紀雅惠] 期刊論文

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