In vitro assay for HCV serine proteinase expressed in insect cells.
OBJECTIVE
To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.


METHODS
The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct 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 and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purified by metal-chelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.


RESULTS
The HCV 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 x 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 its enzymatic activity.


CONCLUSIONS
The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.
Hou
Li-Hua
LH
Department of Applied Molecular Biology, Institute of Microbiology and Epidemiology, Beijing 100071, China. houlihua@sina.com
Du
Gui-Xin
GX
Guan
Rong-Bin
RB
Tong
Yi-Gang
YG
Wang
Hai-Tao
HT
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics*; D066298:In Vitro Techniques; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases