Construction Of HCV-producing Cell Model And Mouse Model Based On Self-cleaving Ribozymes

The hepatitis C virus (HCV) is a major cause of liver disease and infects about 170 million people worldwide, about 3% of the population in the world. In China, more than 50 million people are infected by HCV, and the number is increasing sharply. According to the data reported from China CDC, the newly infected people rose by 20.96% in 2009, reaching 131849. The majority, about 85%, of HCV-infected patients fails to clear the virus, and many develop chronic diseases, including hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). At present, the optimal regimen is a combination of PEG-IFN and ribavirin. This combination therapy results in sustained virological remission (SVR) in only 50% of patients, so it is of necessity to find new antiviral targets, and develop drugs and efficient vaccines, which have been hampered by the lack of robust model systems. The development of the sub genomic and genomic replicon system is a major breakthrough to understand viral replication and viral-host interactions and provides a means to test therapeutic targets. However, as yet, most of these systems can not efficiently produce viral particles, nor do they produce infectious virions. So it is of significance to develop new robust model systems, including in vitro cell models and in vivo animal models.Up to date, all the efficient cells models come from JFH1 system developed by Wakita in 2005. However, its application was limited due to the difficulty to control and operate. The HCV animal models, such as Chimpanzee system and mouse system, progress slowly and work unsatisfactorily.Based on the above situation and urgent need for robust HCV model systems, we constructed an HCV-producing cell model and explored to develop a new mouse model. The research results are as following:1. Preparation of anti-HCV polyclonal antibodies. Six proteins was expressed in E.coli, and then purified. Subsequently, six rabbits were immunized with the recovered proteins respectively. Finally, the blood sera were collected and ELISA was performed to determine the antibody titer.2. Construction of HCV core protein expression vector. To construct the versatile expression vector pEGFP-N1-lac, the lac promoter, which came from pUC19, was inserted into pEGFP-N1 digested with EcoRI/BamHI. Then HCV core gene was amplified by PCR and inserted downstream of the CMV promoter of pEGFP-N1-lac. Western blot analysis showed that this recombinant plasmid can express HCV core protein both in E.coli and in HepG2.3. Construction of HCV-ribozyme plasmid pJFH1/GDD-2RB. The original pJFH1 was reconstructed by putting ribozymes on both 5'and 3'ends of full-length HCV cDNA. Then the fragment containing HCV cDNA and the ribozymes was cloned into pEGFP-N1 to construct target plasmid pJFH1/GDD-2RB. In addition, a mutation in the GDD motif of HCV NS5B, RNA-dependent RNA polymerase (RdRP), was introduced into this construct, and the mutated construct was named pJFH1/GND-2RB as negative control.4. Selection of stable cells lines HepG2/GDD and HepG2/GND. The above constructed plasmids (pJFH1/GDD-2RB and pJFH1/GND-2RB) were transfected into HepG2 cells respectively, and then G418 was added to the final concentration 0.7mg/ml for clone selection. 2 weeks later, the cells without plasmid were dead and the survival cell clones were selected for stably transfected cell line cultivation.5. Determination of HCV RNA in the cell line culture medium. HCV RNA level was quantitated by TaqMan real-time PCR method. HCV RNA was detected in HepG2/GDD culture medium, the level of which was at least 128-fold higher than the level of HepG2/GND culture medium.6. Detection of HCV core protein by immunofluorescence and Western blot analysis. HepG2/GDD cells or negative control cells HepG2 were analyzed by immunofluorescence and Western blot. As expected, the results showed the presence of core protein in HepG2/GDD cells but not in HepG2 cells.7. Detection of HCV particles by electron microscopy. HepG2/GDD cell culture supernatant was precipitated with PEG6000 and put on formavar/carbon-coated copper grids. The grips were negative stained with 2% pphosphotungstic acid (PTA, pH6.5) and observed under a transmission electron microscope. The result showed that these cell culture-derived HCV particles were approximate 55nm in diameter.8. Suppression of HCV replication by IFN-α. To test the sensitivity of HCV production to antiviral, HepG2/GDD cells were treated with IFN-α. 3 days later, HCV RNA was measured in the culture supernatants and the result showed that the HCV RNA level decreased with the increased concentration of IFN-α.9. Transfection of C57 mouse by hydrodynamic injection. A large volume of ribozyme-containing HCV expression plasmid pJFH1/GDD-2RB (10% of the mouse body weight) in a saline solution that is isotonic with blood was injected into mouse blood through the tail vein in less than 10sec. 3 days after injection, the blood serum and liver of C57 were obtained. Real-time RT-PCR showed that HCV RNA was not detectable in the blood, however, the core protein expression was detected in the liver by immunohistochemisty.According to the series of studies shown above, we successfully constructed a HCV-producing cell model based on self-cleaving ribozymes. IFN-treating experiment demonstrated that it can be utilized for anti-HCV drug screening and evaluation. The transfected HCV mouse model was also explored and the probability of using it as an in vivo drug evaluation tool need to be examined.