Establishment and the significance of a cell model of secreted alkaline phosphatase co-controlled by HCV 5'NCR and NS3 serine protease / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 552-553, 2004.
Article
in Chinese
| WPRIM
| ID: wpr-250159
ABSTRACT
<p><b>OBJECTIVE</b>To establish a cell model of secreted alkaline phosphatase (SEAP) co-controlled by HCV 5'NCR and NS3 serine protease in an effort to develop new antiviral agents.</p><p><b>METHODS</b>The fragments of HCV 5'NCR and NS3/4A-SEAP were amplified by PCR. They were fused into pBluescript SK+ to generate 5'NCR-NS3/4A-SEAP chimeric plasmid. The resulting chimeric gene was subcloned into HindIII/Bsu36 I site of pSEAP2-Control (a SEAP eukaryotic expression plasmid), to generate pNCR-NS3/4A-SEAP, in which the SEAP was fused in-frame to the downstream of NS4A/4B cleavage site. The SEAP activity in the culture media of transiently transfected cells was monitored quantitatively. The regulatory effect of HCV 5'NCR and NS3 serine protease on SEAP expression was measured by treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) against HCV 5'NCR and TPCK, a irreversible serine protease inhibitor.</p><p><b>RESULTS</b>The SEAP activity in the culture media reached 80801+/-4794 RLU, and was significantly inhibited by 5 micromol/L, 10 micromol/L of ASODN (t=4.315, p<0.01; t=6.985, p<0.001) and 100 micromol/L of TPCK (t=6.949, P<0.001).</p><p><b>CONCLUSION</b>A cell model of SEAP co-controlled by HCV 5'NCR and NS3 serine protease has been successfully established. This might promote the screening of anti-viral drugs</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Antiviral Agents
/
Virology
/
Recombinant Proteins
/
Serine Endopeptidases
/
Viral Nonstructural Proteins
/
Hepacivirus
/
Bodily Secretions
/
Hepatocytes
/
Drug Evaluation, Preclinical
/
Alkaline Phosphatase
Limits:
Humans
Language:
Chinese
Journal:
Chinese Journal of Hepatology
Year:
2004
Type:
Article
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