[Establishment and the significance of a cell model of secreted alkaline phosphatase co-controlled by HCV 5'NCR and NS3 serine protease].
OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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
Liu
Shui-Ping
SP
Institute of Infectious Diseases, Xiangya Hospital, Central South University, Changsha 410008, China.
Tan
De-Ming
DM
Yang
Yong-Feng
YF
Hou
Zhou-Hua
ZH
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000998:Antiviral Agents; D004353:Drug Evaluation, Preclinical; D016174:Hepacivirus / Q000235:genetics*; D022781:Hepatocytes / Q000201:enzymology / Q000821:virology*; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000469:Alkaline Phosphatase; D000998:Antiviral Agents; D011994:Recombinant 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