AIM:To produce the recombinant NS3 protease of hepatitisC virus with enzymatic activity in insect cells.METHODS:The gene of HCV serine proteinase domainwhich encodes 181 amino acids was inserted intopFastBacHTc and the recombinant plasmid pFBCNS3N wastransformed into DH10Bac competent cells for transposition.After the recombinant bacmids had been determined to becorrect by both blue-white colonies and PCR analysis,theisolated bacmid DNAs were transfected into Sf9 insect cells.The bacmids DNA was verified to replicate in insect cellsand packaged into baculovirus particles via PCR andelectronic microscopic analysis.The insect cells infected withrecombinant baculovirus were determined by SDS-PAGE andWestern-blot assays.The recombinant protein was solutedin N-lauryl sarcosine sodium (NLS) and purifed by metal-chelated-affinity chromatography,then the antigenicity ofrecombinant protease was determined by enzyme-linkedimmunoabsorbant assay and its enzymatic activity wasdetected.RESULTS:The HCV NS3 protease domain was expressedin insect cells at high level and it was partially solved in NLS.Totally 0.2 mg recombinant serine proteinase domain withhigh purity was obtained by metal-chelated-affinitychromatography from 5×10~7 cells,and both antigenicity andspecificity of the protein were evaluated to be high whenused as antigen to detect hepatitis C patients’ sera in indirectELISA format.In vitro cleavage assay corroborated itsenzymatic activity.CONCLUSION:The recombinant HCV NS3 proteinaseexpressed by insect cells is a membrane-binding proteinwith good antigenicity and enzymatic activity. AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells. METHODS: The gene of HCV serine proteinase domainwhich encodes 181 amino acids was inserted intopFastBacHTc and the recombinant plasmid pFBCNS3N wastransformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to becorrect by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells. The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR andelectronic microscopic analysis. insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was solutedin N-lauryl sarcosine sodium (NLS) and purifed by metal-chelated-affinity chromatography, then the antigenic ofrecombinant protease was determined by enzyme-linkedimmunoabsorbant assay and its enzymatic activity wasdetected.RESULTS: The H CV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS. TOTALLY 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5 × 10 ~ 7 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients’ sera in indirect ELISA format. In vitro cleavage assay corroborated itsenzymatic activity. CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.