[Detection of proteolytic activity of hepatitis C virus NS3-4A protease using enzymelinked immunosorbent assay].
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.
Chen
X
X
Department of Virology, Institute of Medicinal Biotechnology, CAMS, PUMC, Beijing 100050, China. xhchen@fhnet.cn.net
Guo
J
J
Chen
H
H
Wanglin
D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000032:analysis* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus