AIM:To establish an efficient,sensitive,cell-based assaysystem for NS3 serine protease in an effort to study furtherthe property of hepatitis C virus (HCV) and develop newantiviral agents.METHODS:We constructed pCI-neo-NS3/4A-SEAP chimericplasmid,in which the secreted alkaline phosphatase (SEAP)was fused in-frame to the downstream of NS4A/4B cleavagesite.The protease activity of NS3 was reflected by the activityof SEAP in the culture media of transient or stable expressioncells.Stably expressing cell lines were obtained by G418selection.Pefabloc SC,a potent irreversible serine proteaseinhibitor,was used to treat the stably expressing cell linesto assess the system for screening NS3 inhibitors.To comparethe activity of serine proteases from 1b and 1a,two chimericclones were constructed and introduced into both transientand stable expression systems.RESULTS:The SEAP activity in the culture media could bedetected in both transient and stable expression systems,and was apparently decreased after Pefabloc SC treatment.In both transient and stable systems,NS3/4A-SEAP chimericgene from HCV genotype lb produced higher SEAP activityin the culture media than that from 1a.CONCLUSION:The cell-based system is efficient andsensitive enough for detection and comparison of NS3protease activity,and screening of anti-NS3 inhibitors.Thefunctional difference between NS3/4A from 1a and 1bsubtypes revealed by this system provides a clue for furtherinvestigations. AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study furtherthe property of hepatitis C virus (HCV) and develop new antiviral agents. METHODS: We constructed pCI-neo- NS3 / 4A- SEAP chimericplasmid , in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A / 4B cleavagesite. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418selection.Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell line sto assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transientand stable expression systems .RESULTS: The SEAP activity in the culture media could bedetected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment.In both transient and stable systems, NS3 / 4A-SEAP chimericgene from HCV genotype lb produced higher SEAP activity in the culture media than that from 1a. CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3 / 4A from 1a and 1bsubtypes revealed by this system provides alue for further investigation.