[Expression of secreted alkaline phosphatase in hepatocytes controlled by HCV IRES].
OBJECTIVE
To establish a cell model of secreted alkaline phosphatase (SEAP) controlled by HCV internal ribosome entry site (IRES).


METHODS
The fragment of HCV 5' noncoding region (5' NCR) was amplified by polymerase chain reaction (PCR), and was immediately cloned the upstream of the SEAP gene of pSEAP2-Control, an SEAP eukaryotic expression plasmid. With the liposome transfection technique, the resulting recombinant plasmid pdNCRSEAP was transfected into hepatocytes QSG7710, and the SEAP activity of cell culture media was monitored quantitatively by the chemiluminescent method. The regulatory effect of the HCV 5' NCR on the SEAP expression was measured by the treatment of transfected cells with antisense oligodeoxynucleotide (ASODN) at 5 mumol and 10 mumol, respectively.


RESULTS
The light emission intensity of pdNCRSEAP expression was 76% that of pSEAP2-Control. The inhibition rates of pNCRSEAP luminescence intensity affected by ASODN of 5 mumol and 10 mumol were 29.2% and 44.6%, respectively, while ASODN had no significant effect on the pSEAP2-Control expression.


CONCLUSIONS
The SEAP expression of pdNCRSEAP is controlled by HCV 5'NCR. The cell model of drug evaluation targeted at HCV 5'NCR is successfully established and can be analyzed conveniently.
Liu
Shui-ping
SP
Institute of Infectious Disease, Xiangya Hospital, Central South University, Changsha 410008, China.
Tan
De-ming
DM
Hou
Zhou-hua
ZH
D020121:5' Untranslated Regions / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004341:Drug Evaluation; D015870:Gene Expression; D016174:Hepacivirus / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology*; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014162:Transfection
D020121:5' Untranslated Regions; D020319:Oligodeoxyribonucleotides, Antisense; D012315:RNA Caps; D012367:RNA, Viral; D000469:Alkaline Phosphatase