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  • Title: [Detection of proteolytic activity of hepatitis C virus NS3-4A protease using enzymelinked immunosorbent assay].
    Author: Chen X, Guo J, Chen H, Wanglin.
    Journal: Zhongguo Yi Xue Ke Xue Yuan Xue Bao; 2000 Jun; 22(3):300-2. PubMed ID: 12903484.
    Abstract:
    OBJECTIVE: To establish an ELISA method for detecting proteolytic activity of HCV serine protease for screening inhibitors against HCV. METHODS: HCV recombinant plasmid pMAL-c2/NS3-4A was transformed into the E. coli strain K12 TB1. Maltose-binding-protein (MBP)-NS3/NS3-4A fusion protein expression were induced by adding isopropyl-beta-D-thiogalacto-pyranoside (IPTG) and purified by affinity chromatography. The immunological activities of the fusion protein were analyzed by Western blot. A peptide substrate was used to analyze the biological activity of the fusion protein. The hydrolyzed product was treated with sodium iodacetate and labeled with digoxigenin, then adding immunological anti-digoxigenin-alkaline phosphatase conjugate to detect the protease activity by colorimeteric reaction. RESULTS: The purified MBP-NS3 and MBP-NS3-4A proteases were identified as 112,000 u and 116,000 u protein by Western blot. HCV NS3/NS3-4A protease showed substrate cleavage activities by ELISA. Coefficients of variation (CV) of ELISA in a lot and among the lots were 4.16% and 7.52% respectively, the P/N was 3.63 under the best experimental conditions determined by L9 (3(4)) factorial design. The method confirmed that 8.36 mumol/L 1,4-naphthoquinone had 50% inhibitive activity on HCV serine protease. CONCLUSIONS: We have established a simple and rapid ELISA method with stable repeatability for detecting proteolytic activities of HCV NS3-4A protease, which might be used for screening and studying of specific inhibitors of HCV serine protease NS3-4A.
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