Establishment Of The Cell Model Expressing Secreted Alkaline Phosphatase Co-controlled By HCV 5'NCR And NS3 Serine Protease And Its Preliminary Application

Objective Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, which has been a worldwide health crisis. Neither efficient chemotherapy nor vaccine protection currently exists. The lack of an in vitro viral replication system and the small animal model of HCV infection hampers anti-HCV studies and drug screening. It is crucial and urgent to develop and apply an efficient and sensitive assay system to select cheap specific anti-HCV ingredients from Chinese medicine. The facts that the highly conserved HCV 5' NCR contains an internal ribosome entry site (IRES) essential for cap-independent translation of viral genes, and that HCV NS3 serine protease is responsible for the maturity of the nonstructural proteins and the viral replication suggest that the HCV 5' NCR and NS3 protease are attractive antiviral targets. Thus we hope to establish a cell model targeted at the HCV 5' NCR and NS3 protease to screen antiviral drugs and to elucidate the antiviral mechanism of oxymatrine and glycyrrhizin. Methods (1) Gene fragments of the HCV 5' NCR and NS3/4A were amplified with PCR. Three recombinant SEAP expression plasmids: 1 pNCRSEAP plasmid expressing secreted alkaline phosphatase (SEAP) controlled by HCV 5'NCR, 2 pNS3 SEAP plasmid expressing SEAP controlled by HCV NS3 serine protease, and 3pNNSEAP plasmid expressing SEAP co-controlled by HCV 5' NCR and NS3 serineprotease were constructed by inserting the HCV 5' NCR, NS3/4A and 5'NCR-NS3/4A fusion fragments immediately upstream the SEAP gene ofpSEAP2-Control, an SEAP eukaryotic expression plasmid, respectively,in which the start codon of SEAP gene was deleted. The structure ofrecombinant plasmids and PCR product was confirmed by restrictionenzyme digestion and DNA sequence analysis. (2) These plasmids weretransfected into hepatocytes QSG7701 with liposome transfectionprotocol, and the activity of SEAP in the cell culture media wasmonitored quantitatively by the chemiluminescent method. Theregulatory effects of the HCV 5' NCR and NS3 serine protease on theSEAP expression were measured by treatment of transfected cells withASODN (5uM, 10uM) and TPCK (0.1 mM) respectively. (3)pNNSEAP-transfected cells were treated with oxymatrine at theconcentration of 0.06, 0.3, 0.6g/L and glycyrrhizin of 0.02, 0.1 g/L and0.2g/L respectively. Their effects on SEAP expression were analyzed.Results (1) The recombinant pNCRNS3/4A plasmid containing the fusion gene of HCV 5' NCR, NS3/4A, and 5' proximal part of SEAP was constructed; (2) The SEAP-expression plasmid pNCRSEAP regulated by HCV 5' NCR was constructed; (3) The SEAP-expression plasmid pNS3SEAP regulated by HCV NS3 serine protease was constructed; (4) The SEAP-expression plasmids pNNSEAP co-regulated by HCV 5' NCR and HCV NS3 serine protease was constructed; (5) All these plasmids showed high SEAP activity in QSG7701 cells; (6) 5 u M and 10 u M of ASODN significantly inhibited the SEAP expression of pNCRSEAP and pNNSEAP (P<0.01), and so did 0.1mM of TPCK (P<0.001); (7) Both of oxymatrine and glycyrrhizin at the applied concentrations showed no cytotoxicity to QSG7701 cells (P>0.05) andno effect on the SEAP expression of pNNSEAP (P>0.05).Conclusions (1) The cell model with the HCV 5' NCR-controlled SEAP expression was successfully established; (2) The cell model with the HCV NS3 serine protease-controlled SEAP expression was successfully established; (3) The cell model with the HCV 5' NCR and NS3 serine protease-co-controlled SEAP expression was also successfully established; (4) These constructed cell models can be applied to the antiviral study and medicine screening targeted at the HCV 5' NCR and/or NS3 serine protease; (5) Oxymatrine and glycyrrhizin had no significant effects on the HCV 5' NCR and NS3 serine protease activity at the applied concentrations. Further experiments should be applied to confirm whether glycyrrhizin has anti-HCV...